Immune profiling by primer extension target enrichment

ABSTRACT

Methods and compositions are described herein for primer extension target enrichment of immune receptor (BCR or TCR) sequences.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation in part of application Ser. No.15/611,507, filed on Jun. 1, 2017, which claims benefit of priority toU.S. Provisional Patent Application No. 62/344,330, filed on Jun. 1,2016, which are incorporated herein by reference for all purposes.

REFERENCE TO A SEQUENCE LISTING

The Sequence Listing written in file SequenceListing_098599-1048987-P33638US1.txt created on Aug. 9, 2017, 77,612bytes, machine format IBM-PC, MS-Windows operating system, is herebyincorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

The adaptive immune system can generate a wide array of diverse bindingmolecules. For example, recombination, random insertion, deletion andsubstitution, has the potential to create between 10¹⁵ and 10²⁰ T-cellreceptor (TCR) clonotypes and considerably more B cell receptor (BCR)clonotypes due to the greater number of VDJ sequences as well as somatichypermutation. Naïve B or T cell clonotypes can be subject to positiveand negative selection pressures, where cells expressing certain immunereceptor sequences are expanded or deleted respectively. As such, therepertoire of BCR or TCR sequences can include information regardingimmunological development, diseases state, the status of an organtransplant (e.g., tolerated or rejected), or the presence or absence ofan autoimmune disorder, cancer, or infection. Moreover, high throughputsequencing of BCR or TCR repertoires has become a powerful tool to studyor monitor basic immunology, disease state, autoimmune disorders,cancer, infection, organ transplantation and the like.

BRIEF SUMMARY OF THE INVENTION

Described herein are improved methods and compositions for primerextension target enrichment (PETE) of immune receptor (TCR or BCR, or acombination thereof) sequences. Methods and compositions describedherein can be used, e.g., for high-throughput sequencing and/or immunerepertoire profiling. In some cases, the methods and compositionsdescribed herein can provide increased sensitivity, decreasedbackground, or increased efficiency in high-throughput sequencinglibrary preparation of immune receptor polynucleotides. In some cases,the methods and compositions described herein can provide increaseddetection of rare immune cell clonotypes, reduced sample requirements,or a combination thereof. In some embodiments, the improvement isprovided by the sequential use of two flanking primers for enrichment ofimmune receptor sequences by PETE, where a first flanking primerhybridizes to a target polynucleotide and is extended with a polymerase,and a second flanking primer hybridizes to the extended first flankingprimer and is extended with a polymerase. Thus, the sample is enrichedfor target polynucleotides containing the first primer hybridizationsite and a complement of the second primer hybridization site.

In a first aspect, the present invention provides a compositioncomprising: i) a plurality of structurally distinct immune cell receptorV gene specific primers, wherein the plurality comprises at least 10structurally distinct primers having the following regions from 5′ to3′: [5′-Phos], [SPLINT], [BARCODE], and [FW], wherein: [5′-Phos]comprises a 5′ phosphate; [SPLINT] comprises an adaptor hybridizationsite of 2-8 nucleotides in length; [BARCODE] comprises a barcode regionof at least 6 nucleotides in length, wherein each nucleotide of thebarcode region is independently selected from the group consisting of Nand W; and [FW] of each immune cell receptor V gene specific primercomprises a structurally distinct region that specifically hybridizes toa framework 1, framework 2, or framework 3 region of an immune cellreceptor V gene.

In some embodiments, [BARCODE] comprises a barcode region of from 6 to16 nucleotides in length. In some embodiments, the [FW] of each immunecell receptor V gene specific primer specifically hybridizes to aframework 1, framework 2, or framework 3 region of a T cell receptor Vgene. In some embodiments, the [FW] of each immune cell receptor V genespecific primer specifically hybridizes to a framework 1, framework 2,or framework 3 region of a B cell receptor V gene. In some embodiments,the plurality comprises at least 10 of the primers set forth in SEQ IDNos:1-121.

In some embodiments, the [SPLINT] consists of 6 consecutive nucleotides,preferably of the sequence CGA TCT. In some embodiments, [BARCODE]consists of thirteen consecutive nucleotides selected from the groupconsisting of N and W, preferably of the sequence WNN NNN WNN NNN W. Insome embodiments, the composition comprises at least 50, preferably allof the primers set forth in SEQ ID NOs:1-121.

In a second aspect, the present invention provides a reaction mixturecomprising: i) a plurality of structurally different targetpolynucleotides, wherein individual target polynucleotides of theplurality each comprise immune cell receptor V gene regions, optionallyD gene regions, optionally C gene regions, and J gene regions; and ii) aplurality of immune cell receptor V gene specific primers according toany one of the preceding aspects or embodiments. In some embodiments,the plurality of immune cell receptor V gene specific primers are eachhybridized to one of the plurality of structurally different targetpolynucleotides.

In some embodiments, a portion of the individual target polynucleotidesof the plurality each comprise immune cell receptor D gene regions and aportion of the individual target polynucleotides of the plurality do notcomprise immune cell receptor D gene regions. In some embodiments, theindividual target polynucleotides of the plurality each comprise immunecell receptor D gene regions. In some embodiments, a portion of theindividual target polynucleotides of the plurality each comprise immunecell receptor C gene regions and a portion of the individual targetpolynucleotides of the plurality do not comprise immune cell receptor Cgene regions. In some embodiments, the individual target polynucleotidesof the plurality each comprise immune cell receptor C gene regions. Insome embodiments, the reaction mixture comprises a plurality of immunecell receptor C gene specific (C-segment) primers. In some embodiments,the plurality of immune cell receptor C gene specific primers arehybridized to one of the structurally different target polynucleotides.

In a third aspect, the present invention provides a reaction mixturecomprising: i) a plurality of immune cell receptor specific first primerextension products, wherein the individual first primer extensionproducts each comprise the following from regions from 5′ to 3′: asequencer-specific adapter sequence, optionally a multiplex identifier(MID) barcode, a unique molecular identifier (UID) barcode, at least aportion of an immune cell receptor framework 3 region, an immune cellreceptor CDR3 region, an optional immune cell receptor diversity (D)region, an optional immune cell receptor constant (C) region, and atleast a portion of an immune cell receptor J region; and ii) a pluralityof J-gene specific primers, wherein each of the plurality of J-genespecific primers is hybridized to the immune cell receptor J region ofone of the individual first primer extension products or a plurality ofC gene specific primers, wherein each of the plurality of C genespecific primers is hybridized to the immune cell receptor C region ofone of the individual first primer extension products.

In some embodiments, the reaction mixture further comprises a DNApolymerase. In some embodiments, the individual first primer extensionproducts each comprise the multiplex identifier (MID) barcode. In someembodiments, a portion of the individual first primer extension productscomprise the immune cell receptor D region and a portion of the firstprimer extension products do not comprise the immune cell receptor Dregion. In some embodiments, the individual target polynucleotides eachcomprise the immune cell receptor D region. In some embodiments, theplurality of J-gene specific primers comprise at least 10 of the primersset forth in SEQ ID Nos:122-204. In some embodiments, the plurality ofJ-gene specific primers comprise at least 50, preferably all of theprimers set forth in SEQ ID Nos: 122-204. In some embodiments, theindividual target polynucleotides each comprise the immune cell receptorC region. In some embodiments, the plurality of C gene specific primerscomprise at least two of the primers set forth in SEQ ID NOs:205-213. Insome embodiments, the plurality of C gene specific primers comprise atleast five, preferably all, of the primers set forth in SEQ IDNOs:205-213.

In a fourth aspect, the present invention provides a reaction mixturecomprising: i) a plurality of first primer extension products, theindividual first primer extension products each comprising the followingfrom 5′ to 3′: a) a 5′ phosphate; b) a SPLINT region comprising anadaptor hybridization site of 2-8 nucleotides in length; c) a uniquemolecular identifier (UID) barcode; d) at least a portion of an immunecell receptor framework 3 region; e) an immune cell receptor CDR3region; f) an optional immune cell receptor diversity region; g) atleast a portion of an immune cell receptor J region; and h) an optionalimmune cell receptor constant region; and i) a plurality ofdouble-stranded splint adapters, each comprising: a) a 5′single-stranded overhang region hybridized to the SPLINT region of anindividual first primer extension product; b) optionally a multiplexidentifier (MID) barcode; and c) a sequencer-specific universal primersequence.

In some embodiments, the reaction mixture further comprises ligase. Insome embodiments, the double-stranded splint adapters each comprise amultiplex identifier (MID) barcode.

In a fifth aspect, the present invention provides a method for enrichingfrom a sample a plurality of structurally different targetpolynucleotides, wherein individual target polynucleotides of theplurality comprise immune cell receptor V, J, and optionally C and/or Dgene regions, the method comprising: a) providing a reaction mixtureaccording to any one of the preceding aspects or embodiments thatprovide a reaction mixture, wherein the immune cell receptor V genespecific primers are hybridized to the V gene regions of the targetpolynucleotides; b) extending the hybridized immune cell receptor V genespecific primers with a polymerase, and then removing un-extended immunecell receptor V gene specific primers, if present, wherein the extendedimmune cell receptor V gene specific primers comprise at least a portionof the immune cell receptor V region, optionally the immune cellreceptor D region, and at least a portion of the immune cell receptor Jregion; c) hybridizing a first universal adaptor to the [SPLINT] adaptorhybridization site of the extended immune cell receptor V gene specificprimers; d) ligating the hybridized first universal adapters to theextended immune cell receptor V gene specific primers, and then removingun-ligated adapters, if present; e) hybridizing a plurality of immunecell receptor J gene specific primers to the J region portions of theextended immune cell receptor V gene specific primers, wherein theimmune cell receptor J gene specific primers comprise a 3′ J genehybridizing region and a 5′ second universal adapter region; and f)extending the hybridized immune cell receptor J gene specific primerswith a polymerase, thereby forming a plurality of structurally differentdouble-stranded products, each comprising at least a portion of theimmune cell receptor V region, optionally the immune cell receptor Dregion, and at least a portion of the immune cell receptor J regionflanked by a first and second universal adapter sequence.

In some embodiments, e) and f) are repeated 2 to 15 times by heating todenature double-stranded products, cooling to hybridize un-extendedimmune cell receptor J gene specific primers to the J region portions ofthe extended immune cell receptor V gene specific primers, and extendinghybridized primers. In some embodiments, the removing un-extended immunecell receptor V gene specific primers comprises digestingsingle-stranded DNA exonuclease digestion. In some embodiments, themethod further comprises amplifying double-stranded products comprisingfirst and second universal adapters by universal PCR. In someembodiments, the structurally different target polynucleotides are cDNA.In some embodiments, prior to step a), a cDNA synthesis step is includedto prepare cDNA from total RNA or mRNA. In some embodiments, step e) ismodified such that the plurality of immune cell receptor J gene specificprimers are substituted for a plurality of immune cell receptor C genespecific primers, thereby forming a plurality of structurally differentdouble-stranded products, each comprising at least a portion of theimmune cell receptor V region and at least a portion of the immune cellreceptor C region.

In some embodiments, the invention is a method for enriching a samplefor a plurality of structurally different target polynucleotidescomprising an immune gene sequence the method comprising: a) contactinga sample with a plurality of immune cell receptor V gene specificprimers, each primer including from 5′ to 3′: [5′-Phos], [SPLINT1],[BARCODE], and [V], wherein: [5′-Phos] is a 5′ phosphate; [SPLINT] is afirst adaptor sequence; [BARCODE] is a unique molecular identifierbarcode; and [V] is a sequence capable of hybridizing to an immune cellreceptor V gene; b) hybridizing and extending the V gene specificprimers to form a plurality of first double-stranded primer extensionproducts; c) contacting the sample with an exonuclease to removeunhybridized V gene specific primers from the first double strandedprimer extension products; d) contacting the sample with a plurality ofimmune cell receptor J gene specific primers, each primer including from5′ to 3′: [5′-Phos], [SPLINT2], and [J], wherein: [5′-Phos] is a 5′phosphate; [SPLINT2] is a second adaptor sequence; and [J] is a sequencecapable of hybridizing to an immune cell receptor J gene; and furthercontacting the sample with a first universal primer capable ofhybridizing to the first adaptor sequence; e) hybridizing and extendingthe J gene specific primers and the first universal primer to form aplurality of second double-stranded primer extension products; f)contacting the sample with an exonuclease to remove unhybridized J genespecific primers and first universal primer from the seconddouble-stranded primer extension products; g) contacting the sample withfirst and second universal primers capable of hybridizing to the firstand second adaptor sequences; h) amplifying the plurality of seconddouble-stranded primer extension products thereby enriching theplurality of structurally different target polynucleotides comprising animmune gene sequence. In some embodiments, the immune genes comprise oneor more of T-cell receptor alpha (TCRA), T-cell receptor beta (TCRB),T-cell receptor gamma (TCRG), T-cell receptor delta (TCRD),Immunoglobulin heavy chain (IGH) and Immunoglobulin light chain lambdaor kappa (IGL and IGK). In some embodiments, the plurality of V genespecific primers and the plurality of J gene specific primers includeprimers from Table 2a

In some embodiments, the hybridizing in steps b) and/or e) comprises oneor more cycles of a step-wise temperature drop of two or more steps, forexample, 20 cycles of temperature change from 60° C. to 57.5° C. and to55° C. In some embodiments, hybridizing and extending in step e)comprises two or more cycles of duplex denaturation, primer annealingand primer extension, for example hybridizing and extending in step e)comprises 10 cycles temperature change from >90° C., to 60° C. to 57.5°C., to 55° C. and to 72° C.

In some embodiments, the first and second universal primers in step g)comprise additional 5′sequences not present in the first and secondadaptors but the first universal primer in step d) does not compriseadditional 5′sequences not present in the first adaptor.

In some embodiments the exonuclease in steps c) and/or f) isthermolabile, e.g., a thermolabile Exonuclease I.

In some embodiments, extending in steps b) and/or e) is with ahigh-fidelity DNA polymerase.

In some embodiments, the method comprises a purification step aftersteps c) and/or h) but not between steps f) and g). In some embodiments,the first and second universal primers comprise a modificationpreventing digestion of the primers with the exonuclease, e.g., aphosphorothioate (PS) bond, a 2′-O-methyl (2′OMe), a 2′-fluoride andInverted ddT. In some embodiments, the V-gene specific primers, theJ-gene specific primers and the first universal primer in step d) do notcomprise a modification preventing digestion of the primers with theexonuclease.

In some embodiments, the method further comprises sequencing theplurality of structurally different target polynucleotides comprising animmune gene sequence.

In some embodiments, the invention is a method for contamination-reducedsequencing a plurality of structurally different target polynucleotidescomprising an immune gene sequence the method comprising: a) contactinga sample with a plurality of immune cell receptor V gene specificprimers, each primer including from 5′ to 3′: [5′-Phos], [SPLINT1],[BARCODE], and [V], wherein: [5′-Phos] is a 5′ phosphate; [SPLINT] is afirst adaptor sequence; [BARCODE] is a unique molecular identifierbarcode (UMI); and [V] is a sequence capable of hybridizing to an immunecell receptor V gene; b) hybridizing and extending the V gene specificprimers to form a plurality of first double-stranded primer extensionproducts; c) contacting the sample with an exonuclease to removeunhybridized V gene specific primers from the first double strandedprimer extension products; d) contacting the sample with a plurality ofimmune cell receptor J gene specific primers, each primer including from5′ to 3′: [5′-Phos], [SPLINT2], and [J], wherein: [5′-Phos] is a 5′phosphate; [SPLINT2] is a second adaptor sequence; and [J] is a sequencecapable of hybridizing to an immune cell receptor J gene; and furthercontacting the sample with a first universal primer capable ofhybridizing to the first adaptor sequence; e) hybridizing and extendingthe J gene specific primers and the first universal primer to form aplurality of second double-stranded primer extension products; f)contacting the sample with an exonuclease to remove unhybridized J genespecific primers and first universal primer from the seconddouble-stranded primer extension products; g) contacting the sample withfirst and second universal primers capable of hybridizing to the firstand second adaptor sequences; h) amplifying the plurality of seconddouble-stranded primer extension products thereby enriching theplurality of structurally different target polynucleotides comprising animmune gene sequence; i) sequencing the plurality of structurallydifferent target polynucleotides comprising an immune gene sequence toobtain a dataset of sequence reads; j) grouping the sequence readshaving an identical UMI into UMI families; k) removing from the datasetUMI families with relative representation of less than 10%, e.g., isless than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1%.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an embodiment of an immuno-PETE reaction. Theembodiment illustrated is a two-primer T cell receptor (TCR) primerextension target enrichment (PETE) reaction.

FIG. 2 is an image of exemplary primer extension products obtained bythe immuno-PETE assay of Example 1 analyzed by agarose gelelectrophoresis.

FIG. 3 is a Venn diagram demonstrating the number of clones and overlapbetween de-duped and de-deduped sequencing data from exemplary primerextension products obtained by the immuno-PETE assay of Example 1.

FIG. 4 illustrates an embodiment of an immuno-PETE reaction. Theembodiment illustrated is a RNA or mRNA based immuno-PETE reaction.

FIG. 5 is an image of exemplary primer extension products obtained bythe immuno-PETE assay of Example 3 analyzed by agarose gelelectrophoresis.

FIG. 6 is a graph showing donotyping of T cell receptor alpha and betachains using human FACS sorted T cells (SEQ ID NO: 274-311).

FIG. 7 is a graph showing donotyping of T cell receptor alpha and betachains using low DNA input amounts from FFPE tissue sample cells (SEQ IDNO:312-317).

FIG. 8 is a diagram of the workflow of the immune-PETE methodillustrating the steps of the first primer extension with a V-geneprimer and removing the excess primer with a thermolabile exonuclease.

FIG. 9 is a diagram of the workflow of the immune-PETE methodillustrating the steps of the second primer extension with a J-geneprimer and a short opposite facing primer and removing the excess ofboth primers with a thermolabile exonuclease.

FIG. 10 is a diagram of the workflow of the immune-PETE methodillustrating the steps of amplifying the primer extension product withtwo universal indexed primers annealing to adapter sites introduced bythe V-gene primer and the J-gene primer.

FIG. 11 is an illustration of the principle of the contaminationreduction method according to the invention.

FIG. 12 shows results of applying the contamination reduction method toan experimental sample.

DEFINITIONS

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art. See, e.g., Lackie, DICTIONARY OF CELL AND MOLECULARBIOLOGY, Elsevier (4^(th) ed. 2007); Sambrook et al., MOLECULAR CLONING,A LABORATORY MANUAL, Cold Spring Harbor Lab Press (Cold Spring Harbor,N.Y. 1989). The term “a” or “an” is intended to mean “one or more.” Theterm “comprise,” and variations thereof such as “comprises” and“comprising,” when preceding the recitation of a step or an element, areintended to mean that the addition of further steps or elements isoptional and not excluded. Any methods, devices and materials similar orequivalent to those described herein can be used in the practice of thisinvention. The following definitions are provided to facilitateunderstanding of certain terms used frequently herein and are not meantto limit the scope of the present disclosure.

As used herein, the term “substantially all” in reference to removingsubstantially all of a component of a reaction mixture means removing atleast 90%, 95%, 99%, or more of a component.

As used herein, the term “immune cell receptor” refers to a T cellreceptor (TCR), or a B cell receptor (BCR) (i.e., antibody). The BCR canbe in a membrane bound form or a secreted form.

“T cell receptor” or “TCR” refers to the antigen recognition complex ofa T cell. The TCR is composed of two different protein chains (e.g.,alpha and beta or gamma and delta). Each chain is composed of twoextracellular domains containing a variable region (V), a joining region(J), and a constant region (C). The variable region containshypervariable complementarity determining regions (CDRs). Beta and deltaTCR chains further contain a diversity region (D) between the V and Jregions. Further TCR diversity is generated by VJ (for alpha and gammachains) and VDJ (for beta and delta chains) recombination. The termsalso refer to various recombinant and heterologous forms, includingsoluble TCRs expressed from a heterologous system.

The B cell receptor or “BCR” refers to the secreted or membrane boundantigen recognition complex of a B cell. The BCR is composed of twodifferent protein chains (e.g., heavy and light). Each chain contains avariable region (V), a joining region (J), and a constant region (C).The variable region contains hypervariable complementarity determiningregions (CDRs). Heavy chains can further contain a diversity region (D)between the V and J regions. Further BCR diversity is generated by VJ(for light chains) and VDJ (for heavy chains) recombination as well assomatic hypermutation of recombined chains. The terms also refer tovarious recombinant and heterologous forms.

As used herein, the term “barcode” refers to a nucleic acid sequencethat can be detected and identified. Barcodes can be 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50 or more nucleotides long. Barcodes can employerror-correcting codes such that one or more errors in synthesis,replication, and/or sequencing can be corrected to identify the barcodesequence. Examples of error correcting codes and their use in barcodesand barcode identification and/or sequencing include, but are notlimited to, those described in U.S. 2010/0,323,348; and U.S. Pat. No.8,715,967. In some cases, the barcodes are designed to have a minimumnumber of distinct nucleotides with respect to all other barcodes of apopulation. The minimum number can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or more. Thus, for example, a population of barcodeshaving a minimum number of at least five distinct nucleotides willdiffer at least five nucleotide positions from all other barcodes in thepopulation.

As used herein, the term “multiplex identifier,” “MID,” and the like,refers to a barcode that identifies a source or sample. As such, all orsubstantially all, MID barcoded polynucleotides from a single source orsample will share an MID of the same sequence; while all, orsubstantially all (e.g., at least 90% or 99%), MID barcodedpolynucleotides from different sources or samples will have a differentMID barcode sequence. Polynucleotides from different sources or samplesand having different MIDs can then be mixed and sequenced in parallelwhile maintaining source/sample information. Thus sequence reads can beassigned to individual samples.

As used herein, the term “universal identifier,” “universal molecularidentifier,” “unique molecular identifier,” “UID,” and the like, refersto a barcode that identifies a polynucleotide to which it is attached.Typically, all, or substantially all (e.g., at least 90% or 99%), UIDbarcodes in a mixture of UID barcoded polynucleotides are unique.

The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleicacids (DNA) or ribonucleic acids (RNA) and polymers thereof in eithersingle- or double-stranded form. Unless specifically limited, the termencompasses nucleic acids containing known analogues of naturalnucleotides that have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. Unless otherwise indicated, a particular nucleicacid sequence also implicitly encompasses conservatively modifiedvariants thereof (e.g., degenerate codon substitutions), alleles,orthologues, SNPs, and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991);Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini etal., Mol. Cell. Probes 8:91-98 (1994)).

“Polymerase” refers to an enzyme that performs template-directedsynthesis of polynucleotides. A DNA polymerase can add free nucleotidesonly to the 3′ end of the newly forming strand. This results inelongation of the newly forming strand in a 5′-3′ direction. No knownDNA polymerase is able to begin a new chain (de novo). DNA polymerasecan add a nucleotide only on to a pre-existing 3′—OH group, and,therefore, needs a primer at which it can add the first nucleotide.Non-limiting examples of polymerases include prokaryotic DNA polymerases(e.g. Pol I, Pol II, Pol III, Pol IV and Pol V), eukaryotic DNApolymerase, telomerase, reverse transcriptase and RNA polymerase.Reverse transcriptase is an RNA-dependent DNA polymerase thatsynthesizes DNA from an RNA template. The reverse transcriptase familycontain both DNA polymerase functionality and RNase H functionality,which degrades RNA base-paired to DNA. RNA polymerase is an enzyme thatsynthesizes RNA using DNA as a template during the process of genetranscription. RNA polymerase polymerizes ribonucleotides at the 3′-endof an RNA transcript.

In some embodiments, a polymerase from the following may be used in apolymerase-mediated primer extension, end-modification (e.g., terminaltransferase, degradation, or polishing), or amplification reaction:archaea (e.g., Thermococcus litoralis (Vent, GenBank: AAA72101),Pyrococcus furiosus (Pfu, GenBank: D12983, BAA02362), Pyrococcus woesii,Pyrococcus GB-D (Deep Vent, GenBank: AAA67131), Thermococcuskodakaraensis KODI (KOD, GenBank: BD175553, BAA06142; Thermococcus sp.strain KOD (Pfx, GenBank: AAE68738)), Thermococcus gorgonarius (Tgo,Pdb: 4699806), Sulfolobus solataricus (GenBank: NC002754, P26811),Aeropyrum pernix (GenBank: BAA81109), Archaeglobus fulgidus (GenBank:029753), Pyrobaculum aerophilum (GenBank: AAL63952), Pyrodictiumoccultum (GenBank: BAA07579, BAA07580), Thermococcus 9 degree Nm(GenBank: AAA88769, Q56366), Thermococcus fumicolans (GenBank: CAA93738,P74918), Thermococcus hydrothermalis (GenBank: CAC18555), Thermococcussp. GE8 (GenBank: CAC12850), Thermococcus sp. JDF-3 (GenBank: AX135456;WO0132887), Thermococcus sp. TY (GenBank: CAA73475), Pyrococcus abyssi(GenBank: P77916), Pyrococcus glycovorans (GenBank: CAC12849),Pyrococcus horikoshii (GenBank: NP 143776), Pyrococcus sp. GE23(GenBank: CAA90887), Pyrococcus sp. ST700 (GenBank: CAC 12847),Thermococcus pacificus (GenBank: AX411312.1), Thermococcus zilligii(GenBank: DQ3366890), Thermococcus aggregans, Thermococcus barossii,Thermococcus celer (GenBank: DD259850.1), Thermococcus profundus(GenBank: E14137), Thermococcus siculi (GenBank: DD259857.1),Thermococcus thioreducens, Thermococcus onnurineus NA1, Sulfolobusacidocaldarium, Sulfolobus tokodaii, Pyrobaculum calidifontis,Pyrobaculum islandicum (GenBank: AAF27815), Methanococcus jannaschii(GenBank: Q58295), Desulforococcus species TOK, Desulfurococcus,Pyrolobus, Pyrodictium, Staphylothermus, Vulcanisaetta, Methanococcus(GenBank: P52025) and other archaeal B polymerases, such as GenBankAAC62712, P956901, BAAA07579)), thermophilic bacteria Thermus species(e.g., flavus, ruber, thermophilus, lacteus, rubens, aquaticus),Bacillus stearothermophilus, Thermotoga maritima, Methanothermusfervidus, KOD polymerase, TNA1 polymerase, Thermococcus sp. 9 degreesN-7, T4, T7, phi29, Pyrococcus furiosus, P. abyssi, T. gorgonarius, T.litoralis, T. zilligii, T. sp. GT, P. sp. GB-D, KOD, Pfu, T.gorgonarius, T. zilligii, T. litoralis and Thermococcus sp. 9N-7polymerases.

In some embodiments, the polymerase can include a RNA polymerase from aneukaryote such as RNA polymerase I, RNA polymerase II, RNA polymeraseIII, RNA polymerase IV or RNA polymerase V. The polymerase can include abacterial RNA polymerase as well as phage and viral RNA polymerases. Insome aspects, the RNA polymerase can include a T7 RNA polymerase, T3 RNApolymerase, K11 RNA polymerase, K1F RNA polymerase, N4 RNA polymerase orSP6 RNA polymerase. In some embodiments, the RNA polymerase (e.g., E.coli RNA polymerase) can include a wild-type, mutant or artificiallyengineered RNA polymerase. As used herein, “an artificially engineeredRNA polymerase” consists, or comprises, of at least one amino acidsubstitution, deletion or insertion with respect to the wild-type ornaturally occurring RNA polymerase from which the artificiallyengineered RNA polymerase is obtained or derived.

In some embodiments, the polymerase can include a reverse transcriptase(RT). RT is an RNA-dependent DNA polymerase that synthesizesdouble-stranded DNA (cDNA) from a single-stranded RNA template. The RTfamily contain both DNA polymerase functionality and RNase Hfunctionality, which degrades RNA base-paired to DNA. RT's arepredominately associated with retroviruses although non-retrovirusesalso use RT (e.g., Hepatitis B virus). In some aspects, the polymerasecan include a RT from Human Immunodeficiency Virus (e.g., HIV-1),Moloney Murine Leukemia Virus (e.g., M-MLV), Human T-Lymphotrophic Virus(e.g., HTLV), Avian Myeloblastosis Virus (e.g., AMV), Rous Sarcoma Virus(e.g., RSV), SuperScript RT (e.g., SuperScript IV), or Telomerase RT(e.g., TERT). In some embodiments, the RT can include a recombinant RT(e.g., an RT having one or more amino acid substitutions, deletions oradditions as compared to the corresponding wild-type or naturallyoccurring RT). In some aspects, the RT includes transcription of RNAfragments of various sizes (e.g., 1 kb, 5 kb, 10 kb, 15 kb, 20 kb, ormore).

The term “thermostable polymerase,” refers to an enzyme that is stableto heat, is heat resistant, and retains sufficient activity to effectsubsequent polynucleotide extension reactions and does not becomeirreversibly denatured (inactivated) when subjected to the elevatedtemperatures for the time necessary to effect denaturation ofdouble-stranded nucleic acids. The heating conditions necessary fornucleic acid denaturation are well known in the art and are exemplifiedin, e.g., U.S. Pat. Nos. 4,683,202, 4,683,195, and 4,965,188, which areincorporated herein by reference. As used herein, a thermostablepolymerase is suitable for use in a temperature cycling reaction such asthe polymerase chain reaction (“PCR”), a primer extension reaction, oran end-modification (e.g., terminal transferase, degradation, orpolishing) reaction. Irreversible denaturation for purposes hereinrefers to permanent and complete loss of enzymatic activity. For athermostable polymerase, enzymatic activity refers to the catalysis ofthe combination of the nucleotides in the proper manner to formpolynucleotide extension products that are complementary to a templatenucleic acid strand. Thermostable DNA polymerases from thermophilicbacteria include, e.g., DNA polymerases from Thermotoga maritima,Thermus aquaticus, Thermus thermophilus, Thermus flavus, Thermusflliformis, Thermus species sps17, Thermus species Z05, Thermuscaldophilus, Bacillus caldotenax, Thermotoga neopolitana, Thermosiphoafricanus, and other thermostable DNA polymerases disclosed above.

In some cases, the nucleic acid (e.g., DNA or RNA) polymerase may be amodified naturally occurring Type A polymerase. A further embodiment ofthe invention generally relates to a method wherein a modified Type Apolymerase, e.g., in a primer extension, end-modification (e.g.,terminal transferase, degradation, or polishing), or amplificationreaction, may be selected from any species of the genus Meiothermus,Thermotoga, or Thermomicrobium. Another embodiment of the inventiongenerally pertains to a method wherein the polymerase, e.g., in a primerextension, end-modification (e.g., terminal transferase, degradation orpolishing), or amplification reaction, may be isolated from any ofThermus aquaticus (Taq), Thermus thermophilus, Thermus caldophilus, orThermus flliformis. A further embodiment of the invention generallyencompasses a method wherein the modified Type A polymerase, e.g., in aprimer extension, end-modification (e.g., terminal transferase,degradation, or polishing), or amplification reaction, may be isolatedfrom Bacillus stearothermophilus, Sphaerobacter thermophilus,Dictoglomus thermophilum, or Escherichia coli. In another embodiment,the invention generally relates to a method wherein the modified Type Apolymerase, e.g., in a primer extension, end-modification (e.g.,terminal transferase, degradation, or polishing), or amplificationreaction, may be a mutant Taq-E507K polymerase. Another embodiment ofthe invention generally pertains to a method wherein a thermostablepolymerase may be used to effect amplification of the target nucleicacid.

As used herein the term “primer” refers to an oligonucleotide that bindsto a specific region of a single stranded template nucleic acid moleculeand initiates nucleic acid synthesis via a polymerase-mediated enzymaticreaction, extending from the 3′ end of the primer and complementary tothe sequence of the template molecule. PCR amplification primers can bereferred to as ‘forward’ and ‘reverse’ primers, one of which iscomplementary to a nucleic acid strand and the other of which iscomplementary to the complement of that strand. Typically, a primercomprises fewer than about 100 nucleotides and preferably comprisesfewer than about 30 nucleotides. Exemplary primers range from about 5 toabout 25 nucleotides. Primers can comprise, for example, RNA and/or DNAbases, as well as non-naturally-occurring bases. The directionality ofthe newly forming strand (the daughter strand) is opposite to thedirection in which DNA polymerase moves along the template strand.

As used herein, the term “universal primer” and “universal primers”refers to a primer that can hybridize to and support amplification oftarget polynucleotides having a shared complementary universal primerbinding site. Similar, the term “universal primer pair” refers to aforward and reverse primer pair that can hybridize to and support PCRamplification of target polynucleotides having shared complementaryforward and reverse universal primer binding sites. Such universalprimer(s) and universal primer binding site(s) can allow single ordouble-primer mediated universal amplification (e.g., universal PCR) oftarget polynucleotide regions of interest.

As used herein the term “sample” refers to any biological sample thatcomprises nucleic acid molecules, typically comprising DNA and/or RNA.Samples may be tissues, cells or extracts thereof, or may be purifiedsamples of nucleic acid molecules. Use of the term “sample” does notnecessarily imply the presence of target sequence within nucleic acidmolecules present in the sample. In some cases, the “sample” comprisesimmune cells (e.g., B cells and/or T cells), or a fraction thereof(e.g., a fraction enriched in genomic DNA, total RNA, or mRNA). In someembodiments, a sample can comprise a FACS sorted population of cells(such as human T cells) or a fixed formalin paraffin embedded (FFPE)tissue sample.

As used herein, the phrase “stringent hybridization conditions” refersto conditions under which a primer/probe will hybridize to its target,typically in a complex mixture of nucleic acids, but not to othernucleic acid sequences present in the complex mixture. Stringentconditions are sequence-dependent and will be different under differentcircumstances. An extensive guide to the hybridization of nucleic acidsis found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, high stringent conditions are selected to be about 5-10° C.lower than the thermal melting point (T_(m)) for the specific sequenceat a defined ionic strength pH. Low stringency conditions are generallyselected to be about 15-30° C. below the T_(m). The T_(m) is thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the primers/probes complementary to thetarget hybridize to the target sequence at equilibrium. Stringentconditions include those in which the salt concentration is less thanabout 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ionconcentration (or other salts) at pH 7.0 to 8.3 and the temperature isat least about 30° C. for short primers/probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long primers/probes (e.g.,greater than 50 nucleotides). Stringent conditions may also be achievedwith the addition of destabilizing agents such as formamide.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention provides a primer extension target enrichment(PETE) method that includes two flanking primers and compositions forperforming and using the flanking primer PETE (FP-PETE) method. The useof two flanking primers increases the stringency of the enrichment stepas compared to methods that require only a single primer or single baitfor enrichment of each structurally distinct target polynucleotide.Thus, in some cases, the FP-PETE method provides improved or synergistictarget enrichment in comparison to other target enrichment methods suchas, e.g., single primer extension target enrichment methods.

Moreover, in contrast to multiplex PCR based methods in which multiplefirst and second amplification primers are in the same reaction mixtureat the same time, the FP-PETE method can include a step of removingun-extended first primers before introducing second primers into areaction mixture. Thus, in some cases, the method can reduce oreliminate competition between first and second primers. As such, in somecases, the first or second primers, or both can be used at significantlyhigher concentrations in the FP-PETE reaction mixture as compared to,e.g., multiplex PCR based methods. Additionally, or alternatively, anincreased number of first or second primers can be used in the FP-PETEreaction mixture as compared to, e.g., multiplex PCR based methods.

The use of a large number of first or second primers, a highconcentration of first or second primers, or a combination thereof, canprovide improved enrichment for, e.g., high-throughput sequencing sampleworkflows in which a large number of different polynucleotide sequencesare targeted and flanking hybridization sequences for the targetsequences are known. Such high-throughput sequencing sample workflowsinclude, but are not limited to, immune repertoire profiling workflowsin which B cell receptor (BCR) or T cell receptor (TCR) sequences areenriched from a sample, sequenced, and analyzed. Flanking primerextension target enrichment methods for immune repertoire profilingworkflows is termed “immuno-PETE.”

Hybridization conditions including those exemplified herein are readilydeterminable by one of ordinary skill in the art, and can includecalculating primer/probe length, salt concentration, and preferentialtemperature to limit non-specific hybridization. In general, longerprimers/probes require higher temperatures for correct annealing, whileshorter primer/probes require lower temperatures. Hybridizationgenerally depends on the ability of denatured DNA to annealcomplementary nucleic acid sequences (e.g., primer/probes) present in anenvironment (e.g., a reaction mixture) below their melting temperature.The higher the degree of homology between a primer/probe and denaturedDNA, the higher the annealing temperature can be while minimizingnon-specific hybridization. Accordingly, higher relative annealingtemperatures tend to make the reaction conditions more “stringent”,while lower annealing temperatures make the reaction conditions less so.Additional details and explanations of hybridization stringency can befound for example in Ausubel et al., (Current Protocols in MolecularBiology, Wiley Interscience Publishers, (1995)).

In some embodiments, the invention is a primer extension targeted geneenrichment assay designed to specifically enrich and amplify humanT-cell receptor (TCR) loci and B-cell receptor (BCR) loci from genomicDNA, and result in unbiased and quantitative TCR and BCR repertoireinformation upon next-generation sequencing analysis. In someembodiments, the invention is a primer extension target enrichment assayoptimized for the human TCR-beta locus (TRB) as well as BCR-heavy chainlocus (IGH). In some embodiments, the invention is a primer extensiontarget enrichment assay optimized for Illumina MiSeq or NextSeqnext-generation sequencing platforms.

In one aspect, the present invention provides first or second primers(e.g., SEQ ID NOs:1-121, 122-204, 205-213, etc.) that selectivelyhybridize to certain target polynucleotides. The phrase “selectively (orspecifically) hybridizes to” refers to the binding, duplexing, orhybridizing of a molecule (e.g., a target polynucleoctide) to aparticular nucleotide sequence (e.g., a primer or probe comprising SEQID NOS:1-213) under stringent hybridization conditions when the targetpolynucleotide is present in a reaction mixture (e.g., total RNA, mRNA,cDNA or gDNA).

In one embodiment, the first or second primers described herein arecomplementary or substantially complementary (i.e., at least 70%, 75%,80%, 85%, 90%, 95% or 99% complementary) to the target polynucleotides.In another embodiment, the first or second primers are complementary orsubstantially complementary (i.e., at least 70%, 75%, 80%, 85%, 90%, 95%or 99% complementary across at least 5, at least 10, at least 15, atleast 20 or more nucleotides) to the target polynucleotides. In anotherembodiment, the first or second primers are complementary across theirfull-length to the target polynucleotides.

In another embodiment, the first or second primers are complementary orsubstantially complementary (i.e., at least 70%, 75%, 80%, 85%, 90%, 95%or 99% complementary) to a framework 1, framework 2, or framework 3region of an immune cell receptor V gene. In another embodiment, thefirst or second primers are complementary or substantially complementary(i.e., at least 70%, 75%, 80%, 85%, 90%, 95% or 99% complementary acrossat least 5, at least 10, at least 15, at least 20 or more nucleotides)to a framework 1, framework 2, or framework 3 region of an immune cellreceptor V gene. In another embodiment, the first or second primers arecomplementary across their full-length to a framework 1, framework 2, orframework 3 region of an immune cell receptor V gene.

In yet another embodiment, the first or second primers are complementaryor substantially complementary (i.e., at least 70%, 75%, 80%, 85%, 90%,95% or 99% complementary) to an immune cell receptor J gene region. Inanother embodiment, the first or second primers are complementary orsubstantially complementary (i.e., at least 70%, 75%, 80%, 85%, 90%, 95%or 99% complementary across at least 5, at least 10, at least 15, atleast 20 or more nucleotides) to an immune cell receptor J gene region.In another embodiment, the first or second primers are complementaryacross their full-length to an immune cell receptor J gene region.

In another embodiment, the first or second primers are complementary orsubstantially complementary (i.e., at least 70%, 75%, 80%, 85%, 90%, 95%or 99% complementary) to an immune cell receptor C gene region. Inanother embodiment, the first or second primers are complementary orsubstantially complementary (i.e., at least 70%, 75%, 80%, 85%, 90%, 95%or 99% complementary across at least 5, at least 10, at least 15, atleast 20 or more nucleotides) to an immune cell receptor C gene region.In another embodiment, the first or second primers are complementaryacross their full-length to an immune cell receptor C gene region.

In some embodiments, genomic DNA or cDNA comprising targetpolynucleotides of the invention can be identified under stringenthybridization conditions using the primer/probes sequences disclosedhere (e.g., comprising any one or more of SEQ ID NOS:1-213). Thefollowing is an exemplary set of hybridization conditions performed on athermocycler and is not limiting: a) denaturation of sample containingtarget polynucleotides at 98° C. for 2 minutes; b) hybridization orprimers/probes at 60° C. for 20 minutes; c) extension of primers/probesusing polymerase at 65° C. for 2 minutes; d) addition of Exonuclease Ito reaction mixture at 37° C. for 10 minutes, increase temperature to80° C. for a further 10 minutes, hold at 4° C.

In one embodiment, target polynucleotides that selectively hybridize toany one of the primer/probe sequences disclosed herein (e.g., SEQ IDNOS: 1-213) can be of any length, e.g., at least 10, 15, 20, 25, 30, 50,100, 200, 500 or more nucleotides or having fewer than 500, 200, 100, or50 nucleotides, etc.).

In one embodiment, a first primer is hybridized to a region of a targetimmune cell receptor polynucleotide that is 3′ to a region of interestor at a 3′ end of a region of interest. For example, the region ofinterest can include at least a portion of the immune cell receptor Vregion, at least a portion of the C region, the diversity (D) region ifpresent, and at least a portion of the J region. In some cases, thefirst primer hybridizes to a framework region (e.g., a framework 1,framework 2, or framework 3 region) of the immune cell receptor Vregion. In some cases, the first primer hybridizes to the immune cellreceptor C region (e.g., comprising a C-segment primer). The firstprimer can then be extended by a polymerase in a first extensionreaction. Un-extended first primers and other single-stranded nucleicacid (e.g., denatured genomic DNA) can then be removed, e.g., bysingle-stranded DNA exonuclease digestion. In a second reaction, asecond primer hybridizes to a region of the extended first primer thatis 3′ to the region of interest or at a 3′ end of a region of interest.Thus, the first and second primers flank the region of interest whenhybridized to their respective targets. In some cases, the second primerhybridizes to a J-gene region of the extended first primer. In somecases, the second primer hybridizes to a V-gene region of the extendedfirst primer. In some cases, the second primer hybridizes to a C generegion of the extended first primer. The second primer can then beextended by a polymerase in a second extension reaction.

Alternatively, a first primer can be hybridized to a J-gene region of atarget immune cell receptor polynucleotide that is 3′ to a region ofinterest or at a 3′ end of a region of interest. The hybridized firstprimer can then be extended by a polymerase. Un-extended first primersand other single-stranded nucleic acid (e.g., denatured genomic DNA) canthen be removed, e.g., by single-stranded DNA exonuclease digestion. Ina second reaction, a second primer can be hybridized to a frameworkregion of the extended first primer and extended with a polymerase.

In one embodiment, complementary DNA (cDNA) can be prepared from RNA ormRNA for use in the PETE methods described herein. In one aspect, cDNAis prepared from total RNA or mRNA isolated and/or purified from a cell,cell lysate, sample or tissue. In one embodiment, RNA can include totalRNA obtained using RNA isolation and/or extraction methods known in theart (e.g., Molecular Cloning: A Laboratory Manual, 4th Edition, Vol. 1,Chapter 6 (2012) incorporated herein by reference for all purposes;RNeasy Mini Kit (Catalog Number: 74104), Qiagen, Germantown, Md.). Inanother embodiment, cDNA can be synthesized from total RNA or mRNAtranscripts using commercially available kits (for example, SuperScriptIII™ Reverse Transcriptase (Catalog Number: 18080093) or SuperScript®VILO™ cDNA Synthesis Kit (Catalog Number: 11754050), ThermoFisherScientific, Waltham, Mass.). In another embodiment, cDNA can be preparedfrom total RNA or mRNA transcripts obtained from whole blood, peripheralblood mononuclear cell (PBMC), sorted lymphocytes, lymphocyte culture,fresh or fresh-frozen tumor tissue or formalin-fixed paraffin embedded(FFPE) tissue (for example, RNeasy FFPE kit, Qiagen, Germantown, Md.).In one aspect, cDNA synthesis can be initiated at or near the 3′ terminiof the mRNA transcript and terminates at or near the 5′ end of the mRNAso as to generate “full-length” cDNA molecules.

In one embodiment, mRNA purified from a sample (e.g., cell, cell lysateor tissue) can be primed with an oligo-dT primer (e.g., a poly-Tprimer), random primer mixture (for example, a mixture or randomhexamers, heptamers, octamers, nanomers, etc.,) or one or moreisotype-specific immune receptor Constant-region (C-segment) primers(e.g., comprising SEQ ID NOS:205-213 of Table 6) under hybridizationconditions sufficient to initiate cDNA synthesis (see FIG. 4). In oneaspect, the oligo-dT primer facilitates the 3′ end of the mRNA's in thesample being represented in the resulting cDNA molecules. In anotheraspect, the one or more isotype-specific immune receptor C-segmentprimers allows for hybridization of the C-segment primer to acomplementary sequence among the one or more mRNA transcripts present inthe sample. In one embodiment, the resulting cDNA molecules can be usedin one or more of the PETE assays as described herein to produce one ormore primer extension products that can be used to identify immunereceptor isotypes in the sample. Accordingly, RNA or mRNA molecules ofthe sample can undergo first, and preferably, second strand synthesis toproduce double-stranded cDNA molecules. In one embodiment, cDNA isprepared using an oligo-dT primer as set forth in this paragraph. Inanother embodiment, cDNA is prepared using a random primer mixture ofhexamers, heptamers, octamers, nanomers, etc., as set forth in thisparagraph. In another embodiment, cDNA is prepared using at least oneisotype-specific immune receptor C-segment primer. In anotherembodiment, cDNA can be prepared using one or more C-gene regionprimers. For example Glanville et al., (PNAS, (2009) 106.48, 20216-21)discloses using a human heavy chain constant region primer, human kappaconstant region primer and human lambda constrant region primer toprepare cDNA from total RNA and/or mRNA from human samples. In yetanother embodiment, cDNA is prepared using at least one of theisotype-specific immune receptor C-segment primers set forth in SEQ IDNOS:205-213.

Once prepared, the synthesized cDNA can be purified by any method knownin the art (e.g., Solid Phase Reversible Immobilization (SPRI)paramagnetic beads or AMPure paramagnetic beads (Beckman Coulter, Brea,Calif.), filtration or centrifugation columns (e.g., RNeasy Mini Kit(Catalog Number: 74104), Qiagen, Germantown, Md.). The purified cDNA canthen serve as a template for any of the methods described herein. In oneembodiment, a plurality of V gene specific primers (e.g., comprising SEQID NOS:1-121) can be used in the first round of primer extension (gPEextension), followed by a second round of extension using one or moreC-segment primers (e.g., comprising SEQ ID NOS:205-213, see Table 6).Alternatively, cDNA prepared as described herein can be used as thestarting template for the PETE assays described herein (e.g., Example1), where a plurality of V gene specific primers (e.g., comprising SEQID NOS:1-121) can be used in the first round of primer extension (gPEextension), followed by a second round of extension (PE2 extension)using a plurality of J-gene specific primers (e.g., comprising SEQ IDNOS:122-204).

II. Compositions

Described herein are compositions for performing immuno-PETE. Suchcompositions can include one or more, or all, of the following: primers,primer sets, polymerase extension products of such primers or primersets (e.g., hybridized to a target polynucleotide), targetpolynucleotides (e.g., single-stranded target polynucleotides), reactionmixtures, DNA-dependent DNA polymerases, RNA-dependent DNA polymerases,single-stranded DNA exonucleases, nucleotides, buffers, salts, and thelike.

In one embodiment, a composition containing a plurality of first primersis provided. In some cases, the plurality of first primers are immunecell receptor gene specific primers. The plurality of immune cellreceptor gene specific primers can be configured to hybridize to, andthus enrich in a polymerase-mediated extension step, a plurality oftarget polynucleotides in a sample that encode immune cell receptorgenes. The first primers can include a 5′-terminal phosphate(“[5′-Phos]”). The 5′-terminal phosphate can allow for ligation of the5′ end of the first primer, or a polymerase extension product thereof,to a 3′-OH of an adjacent polynucleotide.

The first primers can include a [SPLINT] region. The [SPLINT] region caninclude an adapter hybridization site of at least 2 nucleotides inlength. In some cases, the adapter hybridization site is at least 4nucleotides in length, at least 6 nucleotides in length, at least 8nucleotides in length, from 2 to 10 nucleotides in length, or from 2 to8 nucleotides in length. The [SPLINT] region can be complementary to asingle-stranded 5′ overhang region of a double-stranded adapter, suchthat when the single-stranded 5′ overhang region of the double-strandedadapter hybridizes to the [SPLINT] region, a 3′-OH of the adapter can beligated to the [5′-Phos] of the first primer or a polymerase extensionproduct thereof. In some embodiments, [SPLINT] comprises or consists of6 consecutive nucleotides that are complementary to a single-stranded 5′overhang region of a double-stranded adapter. In some embodiments,[SPLINT] comprises or consists of the sequence CGA TCT.

The first primers can include a [BARCODE] region that is or contains abarcode. The [BARCODE] region can be or contain a UID, an MID, or acombination thereof. In some cases, the [BARCODE] region comprises aUID. The [BARCODE] region can be any length from 2 to about 50 or morenucleotides. For UID barcodes, generally, the barcode length andcomposition is selected to encode more sequences than there are uniquetarget polynucleotides to barcode. As such, for immune repertoireprofiling, where estimates of diversity are generally significantlygreater than 10³ and each unique sequence can be represented in a samplemultiple times, the UID barcode can be at least 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or 16 nucleotides in length. For example, in some casesthe barcode can be from 6 to 16 nucleotides in length, from 8 to 14nucleotides in length, or from 10 to 13 nucleotides in length. In someembodiments, the barcode is 13 nucleotides in length. In some cases, thebarcode comprises nucleotides selected from the group consisting of Nand W. In an exemplary embodiment, the barcode has a sequence of WNN NNNWNN NNN W.

The first primers can include a [FW] region. The [FW] region can be orcontain a structurally distinct sequence that specifically hybridizes toa framework 1, framework 2, or framework 3 region of an immune cellreceptor V gene region of an immune cell receptor (TCR or BCR) encodingtarget polynucleotide, wherein upon hybridization to the V gene regionof an immune cell receptor encoding target polynucleotide, the firstprimer can be extended in the direction of the J gene region of theimmune cell receptor encoding target polynucleotide. In someembodiments, the [FW] region hybridizes to a framework 1, framework 2,or framework 3 region of an TCR encoding target polynucleotide. In someembodiments, the [FW] region hybridizes to a framework 1, framework 2,or framework 3 region of a BCR encoding target polynucleotide. Firstprimers containing a [FW] region can be used in an immuno-PETE methodwith second primers that hybridize to the J gene region of an immunecell receptor encoding target polynucleotide. In some embodiments, thefirst primers include the following regions: [5′-Phos], [SPLINT],[BARCODE], and [FW]. In some embodiments, the first primers include thefollowing regions from 5′ to 3′: [5′-Phos], [SPLINT], [BARCODE], and[FW].

Alternatively, the first primers can include a J-specific region ([J])that specifically hybridizes to a J gene region of an immune cellreceptor encoding target polynucleotide, wherein upon hybridization tothe J gene region of an immune cell receptor (TCR or BCR) encodingtarget polynucleotide, the first primer can be extended in the directionof the V gene region of the immune cell receptor encoding targetpolynucleotide. In some embodiments, the [J] region hybridizes to a Jgene region of a TCR encoding target polynucleotide. In someembodiments, the [J] region hybridizes to a J gene region of a BCRencoding target polynucleotide. First primers containing a region [J]can be used in an immuno-PETE method with second primers that hybridizeto the framework 1, framework 2, or framework 3 region of an immune cellreceptor encoding target polynucleotide. In some embodiments, the firstprimers include the following regions: [5′-Phos], [SPLINT], [BARCODE],and [J]. In some embodiments, the first primers include the followingregions from 5′ to 3′: [5′-Phos], [SPLINT], [BARCODE], and [J]. Inanother embodiment, the first primers can include a Constant-specificregion ([C]) that specifically hybridizes to a C gene region of animmune cell receptor encoding target polynucleotide, wherein uponhybridization to the C gene region of an immune cell receptor (TCR orBCR) encoding target polynucleotide, the first primer can be extended ina 3′ direction, through the C gene region of the immune cell receptorencoding target polynucleotide. In some embodiments, the [C] regionhybridizes to a C gene region of a TCR encoding target polynucleotide.In some embodiments, the [C] region hybridizes to a C gene region of aBCR encoding target polynucleotide. In some embodiments, first primerscontaining a C-region [C] can be used in an immuno-PETE method withsecond primers that hybridize to the V-specific region ([V]) thatspecifically hybridizes to a V gene region of an immune cell receptorencoding target polynucleotide. In some embodiments, the first primersinclude the following regions: [5′-Phos], [SPLINT], [BARCODE], and [C].In some embodiments, where the sample includes purified mRNA or totalRNA, the first primers can include a C-segment primer that iscomplementary to, and hybridizes under hybridization conditions with a Cgene region of an immune cell receptor encoding polynucleotide, or acomplement thereof.

In one aspect, the present invention provides first primers (e.g., SEQID NOs: 205-213) that can selectively hybridize to certain targetpolynucleotides. The phrase “selectively (or specifically) hybridizesto” refers to the binding, duplexing, or hybridizing of a molecule(e.g., a target polynucleoctide) to a particular nucleotide sequence(e.g., a primer or probe comprising SEQ ID NOS: 205-213) under stringenthybridization conditions when the target polynucleotide is present in areaction mixture (e.g., total RNA, mRNA, cDNA or gDNA).

As described herein, target polynucleotides encoding a TCR β-chain orδ-chain can contain a D region that is positioned between a V region anda J region, whereas target polynucleotides encoding a TCR α-chain orγ-chain can lack a D region, such that the V region and J region areadjacent. Similarly, target polynucleotides encoding a BCR heavy chaincan contain a D region that is positioned between a V region and a Jregion, whereas target polynucleotides encoding a BCR light chain canlack a D region, such that the V region and J region are adjacent.

As such, first primer extension products of first primers containing an[FW] region that are templated by a TCR β-chain or δ-chain or BCR heavychain can, e.g., contain a VDJ region, or a portion of the V region, a Dregion, and a J region. Similarly, first primer extension products offirst primers containing an [FW] region that are templated by a TCRα-chain or γ-chain or a BCR light chain can lack a D region, and thuscontain a V region or portion thereof adjacent to a J region.Alternatively, first primer extension products of first primerscontaining a [J] region that are templated by a TCR β-chain or δ-chainor BCR heavy chain can, e.g., contain a VDJ region, or a portion of theJ region, a D region, and a V region. Similarly, first primer extensionproducts of first primers containing a [J] region that are templated bya TCR α-chain or γ-chain or a BCR light chain can lack a D region, andthus contain a J region or portion thereof adjacent to a V region.

In some embodiments, the plurality of first primers contains at least 2,at least 5, at least 10, at least 25, at least 50, at least 100, or allof the primers set forth in Table 1 (SEQ ID Nos: 1-121). In oneembodiment, the plurality of first primers contains at least 1 of theprimers set forth in SEQ ID Nos: 1-121. In some embodiments, theplurality of first primers contains at least 2, at least 5, at least 10,or all of the primers set forth in SEQ ID NOS: 205-213. In oneembodiment, the plurality of first primers contains at least 1 of theprimers set forth in SEQ ID NOS: 205-213. In one embodiment, theplurality of first primers are complementary or substantiallycomplementary (i.e., at least 70%, 75%, 80%, 85%, 90%, 95% or 99%complementary) to an immune cell receptor C gene region. In anotherembodiment, the first primers are complementary or substantiallycomplementary (i.e., at least 70%, 75%, 80%, 85%, 90%, 95% or 99%complementary across at least 5, at least 10, at least 15, at least 20or more nucleotides) to an immune cell receptor C gene region. Inanother embodiment, the first primers are complementary across theirfull-length to an immune cell receptor C gene region.

TABLE 1 SEQ ID NO: 1.[5′-Phos]-[SPLINT]-[BARCODE]-GA ATG CCC TGA CAG CTC TCG CTT ATASEQ ID NO: 2.[5′-Phos]-[SPLINT]-[BARCODE]-CT CAG AGA AGT CTG AAA TAT TCG ATG ATCAAT TCT CAG TTG SEQ ID NO: 3.[5′-Phos]-[SPLINT]-[BARCODE]-CC AAA TCG MTT CTC ACC TAA ATC TCC AGACAA AG SEQ ID NO: 4.[5′-Phos]-[SPLINT]-[BARCODE]-CA CCT GAC TCT CCA GAC AAA GCT CATSEQ ID NO: 5. [5′-Phos]-[SPLINT]-[BARCODE]-CC TGA ATG CCC CAA CAG CTC TCSEQ ID NO: 6.[5′-Phos]-[SPLINT]-[BARCODE]-GA TTC TCA GGG CGC CAG TTC TCT ASEQ ID NO: 7.[5′-Phos]-[SPLINT]-[BARCODE]-CC TAA TTG ATT CTC AGC TCA CCA CGT CCA TASEQ ID NO: 8. [5′-Phos]-[SPLINT]-[BARCODE]-TC AGG GCG CCA GTT CCA TGSEQ ID NO: 9.[5′-Phos]-[SPLINT]-[BARCODE]-TC CTA GAT TCT CAG GTC TCC AGT TCC CTASEQ ID NO: 10.[5′-Phos]-[SPLINT]-[BARCODE]-GA GGA AAC TTC CCT GAT CGA TTC TCA GCSEQ ID NO: 11.[5′-Phos]-[SPLINT]-[BARCODE]-CA ACT TCC CTG ATC GAT TCT CAG GTC ASEQ ID NO: 12.[5′-Phos]-[SPLINT]-[BARCODE]-AG GAA ACT TCC CTG ATC AAT TCT CAG GTC ASEQ ID NO: 13.[5′-Phos]-[SPLINT]-[BARCODE]-GG AAA CTT CCC TCC TAG ATT TTC AGG TCGSEQ ID NO: 14.[5′-Phos]-[SPLINT]-[BARCODE]-CC CCA ATG GCT ACA ATG TCT CCA GAT TSEQ ID NO: 15.[5′-Phos]-[SPLINT]-[BARCODE]-GG AGA GGT CCC TGA TGG CTA CAASEQ ID NO: 16.[5′-Phos]-[SPLINT]-[BARCODE]-TC CCT GAT GGT TAT AGT GTC TCC AGA GCSEQ ID NO: 17.[5′-Phos]-[SPLINT]-[BARCODE]-GG AGA AGT CCC CAA TGG CTA CAA TGT CSEQ ID NO: 18.[5′-Phos]-[SPLINT]-[BARCODE]-AA AGG AGA AGT CCC GAA TGG CTA CAASEQ ID NO: 19.[5′-Phos]-[SPLINT]-[BARCODE]-GT TCC CAA TGG CTA CAA TGT CTC CAG ATCSEQ ID NO: 20.[5′-Phos]-[SPLINT]-[BARCODE]-GA AGT CCC CAA TGG CTA CAA TGT CTC TAG ATTSEQ ID NO: 21.[5′-Phos]-[SPLINT]-[BARCODE]-GA GAA GTC CCC GAT GGC TAC AAT GTASEQ ID NO: 22.[5′-Phos]-[SPLINT]-[BARCODE]-GT GAT CGG TTC TCT GCA CAG AGG TSEQ ID NO: 23.[5′-Phos]-[SPLINT]-[BARCODE]-CG CTT CTC TGC AGA GAG GAC TGGSEQ ID NO: 24.[5′-Phos]-[SPLINT]-[BARCODE]-GG TTC TTT GCA GTC AGG CCT GASEQ ID NO: 25.[5′-Phos]-[SPLINT]-[BARCODE]-CA GTG GTC GGT TCT CTG CAG AGSEQ ID NO: 26.[5′-Phos]-[SPLINT]-[BARCODE]-GC TCA GTG ATC AAT TCT CCA CAG AGA GGTSEQ ID NO: 27.[5′-Phos]-[SPLINT]-[BARCODE]-TT CTC TGC AGA GAG GCC TGA GGSEQ ID NO: 28.[5′-Phos]-[SPLINT]-[BARCODE]-CC CAG TGA TCG CTT CTT TGC AGA AASEQ ID NO: 29.[5′-Phos]-[SPLINT]-[BARCODE]-CT GCA GAG AGG CCT AAG GGA TCTSEQ ID NO: 30.[5′-Phos]-[SPLINT]-[BARCODE]-GA AGG GTA CAA TGT CTC TGG AAA CAA ACTCAA G SEQ ID NO: 31.[5′-Phos]-[SPLINT]-[BARCODE]-GG GGT ACT GTG TTT CTT GAA ACA AGC TTG AGSEQ ID NO: 32.[5′-Phos]-[SPLINT]-[BARCODE]-CA GTT CCC TGA CTT GCA CTC TGA ACT AAA CSEQ ID NO: 33.[5′-Phos]-[SPLINT]-[BARCODE]-AC TAA CAA AGG AGA AGT CTC AGA TGG CTA CAGSEQ ID NO: 34.[5′-Phos]-[SPLINT]-[BARCODE]-AG ATA AAG GAG AAG TCC CCG ATG GCT ASEQ ID NO: 35.[5′-Phos]-[SPLINT]-[BARCODE]-GA TAC TGA CAA AGG AGA AGT CTC AGA TGGCTA TAG SEQ ID NO: 36.[5′-Phos]-[SPLINT]-[BARCODE]-CT AAG GAT CGA TTT TCT GCA GAG AGG CTCSEQ ID NO: 37.[5′-Phos]-[SPLINT]-[BARCODE]-TT GAT TCT CAG CAC AGA TGC CTG ATG TSEQ ID NO: 38.[5′-Phos]-[SPLINT]-[BARCODE]-AT TCT CAG CTG AGA GGC CTG ATG GSEQ ID NO: 39.[5′-Phos]-[SPLINT]-[BARCODE]-GG ATC GAT TCT CAG CTA AGA TGC CTA ATG CSEQ ID NO: 40.[5′-Phos]-[SPLINT]-[BARCODE]-CT CAG CAG AGA TGC CTG ATG CAA CTT TASEQ ID NO: 41.[5′-Phos]-[SPLINT]-[BARCODE]-CT GAT CGA TTC TCA GCT CAA CAG TTC AGTSEQ ID NO: 42.[5′-Phos]-[SPLINT]-[BARCODE]-TA GCT GAA AGG ACT GGA GGG ACG TATSEQ ID NO: 43.[5′-Phos]-[SPLINT]-[BARCODE]-CC AGG AGG CCG AAC ACT TCT TTC TSEQ ID NO: 44.[5′-Phos]-[SPLINT]-[BARCODE]-GC TAA GTG CCT CCC AAA TTC ACC CTSEQ ID NO: 45.[5′-Phos]-[SPLINT]-[BARCODE]-CA CAG CTG AAA GAC CTA ACG GAA CGTSEQ ID NO: 46.[5′-Phos]-[SPLINT]-[BARCODE]-CT GCT GAA TTT CCC AAA GAG GGC CSEQ ID NO: 47. [5′-Phos]-[SPLINT]-[BARCODE]-AG GGT ACA GCG TCT CTC GGGSEQ ID NO: 48.[5′-Phos]-[SPLINT]-[BARCODE]-GC CTG ACC TTG TCC ACT CTG ACASEQ ID NO: 49.[5′-Phos]-[SPLINT]-[BARCODE]-AT GAG CGA TTT TTA GCC CAA TGC TCC ASEQ ID NO: 50.[5′-Phos]-[SPLINT]-[BARCODE]-TG AAG GCT ACG TGT CTG CCA AGA GSEQ ID NO: 51.[5′-Phos]-[SPLINT]-[BARCODE]-CT CAT CTC AAT GCC CCA AGA ACG CSEQ ID NO: 52.[5′-Phos]-[SPLINT]-[BARCODE]-AG ATC TCT GAT GGA TAC AGT GTC TCT CGA CASEQ ID NO: 53.[5′-Phos]-[SPLINT]-[BARCODE]-AG ATC TTT CCT CTG AGT CAA CAG TCT CCAGAA TA SEQ ID NO: 54.[5′-Phos]-[SPLINT]-[BARCODE]-CA CTG AAA AAG GAG ATA TCT CTG AGG GGTATC ATG SEQ ID NO: 55.[5′-Phos]-[SPLINT]-[BARCODE]-GT TCC TGA AGG GTA CAA AGT CTC TCG AAA AGSEQ ID NO: 56.[5′-Phos]-[SPLINT]-[BARCODE]-CT GAG GGG TAC AGT GTC TCT AGA GAG ASEQ ID NO: 57.[5′-Phos]-[SPLINT]-[BARCODE]-AG CCG CCC AAA CCT AAC ATT CTC AASEQ ID NO: 58. [5′-Phos]-[SPLINT]-[BARCODE]-CC CAG GAC CGG CAG TTC ASEQ ID NO: 59.[5′-Phos]-[SPLINT]-[BARCODE]-TT GAT TAG AGA CAT ATC CCT ATT GAA AAT ATTTCC TGG CA SEQ ID NO: 60.[5′-Phos]-[SPLINT]-[BARCODE]-AG ATG CCC TGA GTC AGC ATA GTC ATT CTA ACSEQ ID NO: 61.[5′-Phos]-[SPLINT]-[BARCODE]-GG AGG GGA AGG CCC CAC AGC GTC TTCSEQ ID NO: 62.[5′-Phos]-[SPLINT]-[BARCODE]-TG AAG TCA TAC AGT TCC TGG TGT CCA TSEQ ID NO: 63.[5′-Phos]-[SPLINT]-[BARCODE]-CC AAA TCA GGC TTT GGA GCA CCT GAT CTSEQ ID NO: 64.[5′-Phos]-[SPLINT]-[BARCODE]-CC AAA CAA AGG CTT AGA ATA TTT ATT ACATGT C SEQ ID NO: 65.[5′-Phos]-[SPLINT]-[BARCODE]-CC AGG TCC CTG AGG CAC TCC ACC AGC TSEQ ID NO: 66.[5′-Phos]-[SPLINT]-[BARCODE]-CT GAA TCT AAA TTA TGA GCC ATC TGA CASEQ ID NO: 67.[5′-Phos]-[SPLINT]-[BARCODE]-TC ATT CCT TAG TCG CTC TGA TAG TTA TGG TTASEQ ID NO: 68.[5′-Phos]-[SPLINT]-[BARCODE]-CA TTC CTT AGT CGG TCT AAA GGG TAC AGT TASEQ ID NO: 69.[5′-Phos]-[SPLINT]-[BARCODE]-AC AAC ATG ACC TAT GAA CGG TTC TCT TCA TCSEQ ID NO: 70.[5′-Phos]-[SPLINT]-[BARCODE]-CT GAA TTT AAC AAG AGC CAA ACC TCC TTC CASEQ ID NO: 71.[5′-Phos]-[SPLINT]-[BARCODE]-CC GAC AGA AAG TCC AGC ACT CTG AGSEQ ID NO: 72.[5′-Phos]-[SPLINT]-[BARCODE]-CA CTG TTC TAT TGA ATA AAA AGG ATA AACATC TGT C SEQ ID NO: 73.[5′-Phos]-[SPLINT]-[BARCODE]-GT CAC CTT TGA TAC CAC CCT TAA ACA GAG TTTSEQ ID NO: 74.[5′-Phos]-[SPLINT]-[BARCODE]-AG ACT AAA TGC TAC ATT ACT GAA GAA TGGAAG CAG SEQ ID NO: 75.[5′-Phos]-[SPLINT]-[BARCODE]-TG AGG CTG AAT TTA TAA AGA GTA AAT TCTCCT TTA A SEQ ID NO: 76.[5′-Phos]-[SPLINT]-[BARCODE]-GC TGA ATT TAA GAA GAG TGA AAC CTC CTT CCASEQ ID NO: 77.[5′-Phos]-[SPLINT]-[BARCODE]-GG CTG AAT TTA AGA GGA GTC AAT CTT CCTTCA A SEQ ID NO: 78.[5′-Phos]-[SPLINT]-[BARCODE]-GA CAC TTA TCA CTT CCC CAA TCA ATA CCC CSEQ ID NO: 79.[5′-Phos]-[SPLINT]-[BARCODE]-GG CTG AAT TTA ACA AGA GTC AAA CTT CCT TCCASEQ ID NO: 80.[5′-Phos]-[SPLINT]-[BARCODE]-GC TGA ATT TAA GAA GAG CGA AAC CTC CTT CTASEQ ID NO: 81.[5′-Phos]-[SPLINT]-[BARCODE]-CC ATG TAC CGT AAA GAA ACC ACT TCT TTC CASEQ ID NO: 82.[5′-Phos]-[SPLINT]-[BARCODE]-CC ACA TAC CGT AAA GAA ACC ACT TCT TTC CASEQ ID NO: 83.[5′-Phos]-[SPLINT]-[BARCODE]-TG GAT GCA GAC ACA AAG CAA AGC TCSEQ ID NO: 84.[5′-Phos]-[SPLINT]-[BARCODE]-TA AAG AAC TGC TTG GAA AAG AAA AAT TTTATA GTG T SEQ ID NO: 85.[5′-Phos]-[SPLINT]-[BARCODE]-AC AGC TCA ATA GAG CCA GCC AGT ATA TTT CSEQ ID NO: 86.[5′-Phos]-[SPLINT]-[BARCODE]-CA GCT CAA TAA AGC CAG CCA GTA TGT TTCSEQ ID NO: 87.[5′-Phos]-[SPLINT]-[BARCODE]-GC ACA GGT CGA TAA ATC CAG CAA GTA TAT CTCSEQ ID NO: 88.[5′-Phos]-[SPLINT]-[BARCODE]-CT GTT ACA TTG AAC AAG ACA GCC AAA CATTTC TC SEQ ID NO: 89.[5′-Phos]-[SPLINT]-[BARCODE]-CA CCG TTT TAT TGA ATA AGA CAG TGA AACATC TCT C SEQ ID NO: 90.[5′-Phos]-[SPLINT]-[BARCODE]-CC AGA AGG CAA GAA AAT CCG CCA ASEQ ID NO: 91.[5′-Phos]-[SPLINT]-[BARCODE]-AG AAG CGC TTG GAA AAG AGA AGT TTT ATAGTG T SEQ ID NO: 92.[5′-Phos]-[SPLINT]-[BARCODE]-TG ACC TTA ACA AAG GCG AGA CAT CTT TCC ASEQ ID NO: 93.[5′-Phos]-[SPLINT]-[BARCODE]-CG CTT GAC ACT TCC AAG AAA AGC AGT TCSEQ ID NO: 94.[5′-Phos]-[SPLINT]-[BARCODE]-CA GTC CTA TCA AGA GTG ACA GTT CCT TCC ASEQ ID NO: 95.[5′-Phos]-[SPLINT]-[BARCODE]-GA ACT TCC AGA AAT CCA CCA GTT CCT TCA ASEQ ID NO: 96.[5′-Phos]-[SPLINT]-[BARCODE]-GC TAA AAG CCA CAT TAA CAA AGA AGG AAAGCT T SEQ ID NO: 97.[5′-Phos]-[SPLINT]-[BARCODE]-CT CGC TGG ATA AAT CAT CAG GAC GTA GTA CSEQ ID NO: 98.[5′-Phos]-[SPLINT]-[BARCODE]-TC GCT ACG GAA CGC TAC AGC TTSEQ ID NO: 99.[5′-Phos]-[SPLINT]-[BARCODE]-CT CCT TCA ATA AAA GTG CCA AGC AGT TCT CSEQ ID NO:[5′-Phos]-[SPLINT]-[BARCODE]-CA CTC TTA ATA CCA AGG AGG GTT ACA GCT A100. SEQ ID NO:[5′-Phos]-[SPLINT]-[BARCODE]--TC AGT TTG GAG AAG CAA AAA AGA ACA GCT C101. SEQ ID NO:[5′-Phos]-[SPLINT]-[BARCODE]-GA TCA TCA CAG AAG ACA GAA AGT CCA GCA C102. SEQ ID NO:[5′-Phos]-[SPLINT]-[BARCODE]-GC AAT CGC TGA AGA CAG AAA GTC CAG TAC 103.SEQ ID NO:[5′-Phos]-[SPLINT]-[BARCODE]-CA GTT TGG TGA TGC AAG AAA GGA CAG TTC 104.SEQ ID NO:[5′-Phos]-[SPLINT]-[BARCODE]-CA GTC AAA GCT GAG GAA CTT TAT GGC CA 105.SEQ ID NO:[5′-Phos]-[SPLINT]-[BARCODE]-CT TCT TAA ACA AAA GTG CCA AGC ACC TCT C106. SEQ ID NO:[5′-Phos]-[SPLINT]-[BARCODE]--CT GCT TCA TTT AAT GAA AAA AAG CAG CAA107. AGC TC SEQ ID NO:[5′-Phos]-[SPLINT]-[BARCODE]-TT CTG TGA GCT TCC AGA AAA CAA CTA AAA 108.CTA TTC A SEQ ID NO:[5′-Phos]-[SPLINT]-[BARCODE]-CA CTG TAC TGT TGA ATA AAA ATG CTA AAC 109.ATG TCT C SEQ ID NO:[5′-Phos]-[SPLINT]-[BARCODE]--GC CTG TGA ACT TTG AAA AAA AGA AAA AGT110. TCA TCA A SEQ ID NO:[5′-Phos]-[SPLINT]-[BARCODE]--CC AAG TTG GAT GAG AAA AAG CAG CAA AGT TC111. SEQ ID NO:[5′-Phos]-[SPLINT]-[BARCODE]--TC AGT TTG GTA TAA CCA GAA AGG ACA GCT T112. SEQ ID NO:[5′-Phos]-[SPLINT]-[BARCODE]--AA GTA GCA TAT TAG ATA AGA AAG AAC TTT113. CCA GCA T SEQ ID NO:[5′-Phos]-[SPLINT]-[BARCODE]-CA GGC TTA AAA AAG GAG ACC AGC ACA TTT C114. SEQ ID NO:[5′-Phos]-[SPLINT]-[BARCODE]-CT TCC AGA AAG CAG CCA AAT CCT TCA G 115.SEQ ID NO: [5′-Phos]-[SPLINT]-[BARCODE]-TG ATA CCA AAG CCC GTC TCA GCA C116. SEQ ID NO:[5′-Phos]-[SPLINT]-[BARCODE]--GA GGC GGA AAT ATT AAA GAC AAA AAC TCC CC117. SEQ ID NO:[5′-Phos]-[SPLINT]-[BARCODE]--CC ACA ATA AAC ATA CAG GAA AAG CAC AGC TC118. SEQ ID NO:[5′-Phos]-[SPLINT]-[BARCODE]-AG AAA GCA GCG AAA TCC GTC GC 119.SEQ ID NO:[5′-Phos]-[SPLINT]-[BARCODE]-TG ACA TTG ATA TTG CAA AGA ACC TGG CTG T120. SEQ ID NO:[5′-Phos]-[SPLINT]-[BARCODE]-GA AAC ACA TTC TGA CCC AGA AAG CCT TTC A121.

In one embodiment, a composition containing a plurality of secondprimers is provided. In some cases, the plurality of second primers areimmune cell receptor gene-specific primers. The plurality of immune cellreceptor gene-specific primers can be configured to hybridize to, andthus enrich in a polymerase-mediated extension step, a plurality oftarget polynucleotides in a sample that encode immune cell receptorgenes. The second primers can include a universal primer binding site,or complement thereof, and an immune cell receptor hybridizing region.In some cases, the second primers include from 5′ to 3′ a universalprimer binding site and an immune cell receptor hybridizing region. Insome cases, the second primer immune cell receptor hybridizing region iscomplementary to, and hybridizes under hybridization conditions with, aJ-gene region of an immune cell receptor encoding polynucleotide, or acomplement thereof. In some cases, the second primer immune cellreceptor hybridizing region is complementary to, and hybridizes underhybridization conditions with, a V region of an immune cell receptorencoding polynucleotide (e.g., a framework 1, framework 2, or framework3 region), or a complement thereof. In some cases, the second primerimmune cell receptor hybridizing region is complementary to, andhybridizes under hybridization conditions with, a C gene region of animmune cell receptor encoding polynucleotide, or a complement thereof.

As described herein, target polynucleotides encoding a TCR β-chain orδ-chain can contain a D region that is positioned between a V region anda J region, whereas target polynucleotides encoding a TCR α-chain orγ-chain can lack a D region, such that the V region and J region areadjacent. Similarly, target polynucleotides encoding a BCR heavy chaincan contain a D region that is positioned between a V region and a Jregion, whereas target polynucleotides encoding a BCR light chain canlack a D region, such that the V region and J region are adjacent.

Second primer extension products of second primers containing a [J] oran [FW] region that are templated by a first primer extension producttemplated by a TCR β-chain or δ-chain or BCR heavy chain can, e.g.,contain a VDJ region, or a portion of the V region, a D region, and aportion of a J region. Similarly, second primer extension products ofsecond primers containing a [J] or an [FW] region that are templated byfirst primer extension product templated by a TCR α-chain or γ-chain ora BCR light chain can lack a D region, and thus contain a V region orportion thereof adjacent to a J region or portion thereof.

In some embodiments, the plurality of second primers contains at least2, at least 5, at least 10, at least 25, at least 50, at least 75, orall of the primers set forth in Table 2 (SEQ ID Nos: 122-204. In oneembodiment, the plurality of second primers contains at least 1 of theprimers set forth in SEQ ID Nos: 122-204. In some embodiments, theplurality of second primers contains at least 2, at least 5, at least10, or all of the primers set forth in SEQ ID NOS: 205-213. In oneembodiment, the plurality of second primers contains at least 1 of theprimers set forth in SEQ ID NOS: 205-213.

In one embodiment, the second primers are complementary or substantiallycomplementary (i.e., at least 70%, 75%, 80%, 85%, 90%, 95% or 99%complementary) to a framework 1, framework 2, or framework 3 region ofan immune cell receptor V gene. In another embodiment, the secondprimers are complementary or substantially complementary (i.e., at least70%, 75%, 80%, 85%, 90%, 95% or 99% complementary across at least 5, atleast 10, at least 15, at least 20 or more nucleotides) to a framework1, framework 2, or framework 3 region of an immune cell receptor V gene.In another embodiment, the second primers are complementary across theirfull-length to a framework 1, framework 2, or framework 3 region of animmune cell receptor V gene.

In yet another embodiment, the second primers are complementary orsubstantially complementary (i.e., at least 70%, 75%, 80%, 85%, 90%, 95%or 99% complementary) to an immune cell receptor J gene region. Inanother embodiment, the second primers are complementary orsubstantially complementary (i.e., at least 70%, 75%, 80%, 85%, 90%, 95%or 99% complementary across at least 5, at least 10, at least 15, atleast 20 or more nucleotides) to an immune cell receptor J gene region.In another embodiment, the second primers are complementary across theirfull-length to an immune cell receptor J gene region.

TABLE 2 SEQ ID NO: Sequence SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 122. ATC TGG GGA GAA GTG GAA ACT CTG GTT CC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 123. ATC TGA CTC ACC AGA TAT AAT GAA TAC ATG GGT CCC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 124. ATC TCC GGA TGC TGA GTC TGG TCC C SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 125. ATC TTG TAC AGC CAG CCT GGT CCC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 126. ATC TCT TGG TTG CAC TTG GAG TCT TGT TCC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 127. ATC TCA CGG ATG AAC AAT AAG GCT GGT TCC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 128. ATC TTT GGT ATG ACC ACC ACT TGG TTC CC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 129. ATC TAC TGA CCA GAA GTC GGG TGC C SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 130. ATC TAT GGA ACT TAC TTG CTT TAA CAA ATA GTC TTG TTC C SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 131. ATC TAC TGA GTT CCA CTT TTA GCT GAG TGC C SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 132. ATC TAC CTG GAG AGA CTA GAA GCA TAG TCC C SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 133. ATC TCC TGA CCA GCA GTC TGG TCC C SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 134. ATC TCT TGG GAT GAC TTG GAG CTT TGT TCC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 135. ATC TCC TAC TTA CCA GGT TTT ACT GAT AAT CTT GTC CC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 136. ATC TTA CTG GAA CTC ACT GAT AAG GTG GTT CC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 137. ATC TCT GGA ACT CAC TGA TAA GGT GGG TTC C SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 138. ATC TTA CTT ACT AAG ATC CAC CTT TAA CAT GGT TCC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 139. ATC TAC TTG GTT TAA CTA GCA CCC TGG TTC C SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 140. ATC TAG GCC AGA CAG TCA ACT GAG TTC C SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 141. ATC TCT CAC TTA CTT GGA GTG ACA TTA TGT TTG GAT CC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 142. ATC TTA CTT ACT TGC TCT TAC AGT TAC TGT GGT TCC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 143. ATC TTA ACT TAC TTG GTT TTA CAT TGA GTT TGG TCC C SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 144. ATC TTA CCA GGT AAA ACA GTC AAT TGT GTC CC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 145. ATC TAC TGG GTT TCA CAG ATA ACT CCG TTC C SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 146. ATC TGG TGA CCA CAA CCT GGG TCC C SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 147. ATC TGC TTG ACA AGC AGC CTT GTC CC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 148. ATC TGC AGC ACG GAC AAT CTG GTT CC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 149. ATC TGG CTT CAC AGT GAG CGT AGT CCC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 150. ATC TCT TGG TAT GAC CGA GAG TTT GGT CCC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 151. ATC TAC TTG CAA TCA CAG AAA GTC TTG TGC C SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 152. ATC TCT GGG GAG AAT ATG AAG TCG TGT CCC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 153. ATC TGC TTC ACC ACC AGC TGA GTT CC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 154. ATC TCT GGA CAG CAA GCA GAG TGC C SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 155. ATC TGA CTT ACC TGG CTT TAT AAT TAG CTT GGT CCC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 156. ATC TTA CTT ACT TGG AAA GAC TTG TAA TCT GGT CCC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 157. ATC TCT TAC GTG GTA AAA CAA TCA CTT GAG TGC C SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 158. ATC TGG GGA ATA ACG GTG AGT CTC GTT CC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 159. ATC TCC TAC CTG GTT TTA CTT GGT AAA GTT GTC CC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 160. ATC TCG GAT TTA CTG CCA GGC TTG TTC C SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 161. ATC TCG GGG TTT GAC CAT TAA CCT TGT TCC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 162. ATC TGC TAA AAC CTT CAG CCT GGT GCC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 163. ATC TGT GAC CAA CAG CGA GGT GCC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 164. ATC TAC ACT TAC TTG GTT TAA CAG AGA GTT TAG TGC C SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 165. ATC TAC TTG GTT TTA CTG TCA GTC TGG TCC C SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 166. ATC TCG AGC GTG ACC TGA AGT CTT GTT CC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 167. ATC TCA GGG CTG GAT GAT TAG ATG AGT CCC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 168. ATC TGG GCC TAA CTG CTA AAC GAG TCC C SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 169. ATC TCA CAG GAC TTG ACT CTC AGA ATG GTT CC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 170. ATC TAC TGG GTA TGA TGG TGA GTC TTG TTC C SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 171. ATC TCT TGG AAT GAC CGT CAA ACT TGT CCC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 172. ATC TAC TTA CTT GGA ATG ACT GAT AAG CTT GTC CC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 173. ATC TTG CTT GGC TTC ACA GTT AGT CAT GTC TC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 174. ATC TCT TGG ATG GAC AGT CAA GAT GGT CCC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 175. ATC TAA CTT ACT TGG ATT CAC GGT TAA GAG AGT TCC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 176. ATC TTT GGG TTG ATA GTC AGC CTG GTT CC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 177. ATC TCC ACA CTT ACT TGG ATT TAT TTT TGT ACT CAT CCC C SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 178. ATC TTA AAA CAT ACC TGG TCT AAC ACT CAG AGT TAT TCC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 179. ATC TTA CAT GGG TTT ACT GTC AGT TTC GTT CC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 180. ATC TCC AGG ATT CAC TGT GAG CTG TGT TCC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 181. ATC TAG CTT CAC TCT CAC TTG CGT CCC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 182. ATC TCC CAG GCT CAC AAT TAA CTC AGT CCC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 183. ATC TTA CTT GCT GAG TTT CAT GAT TCC TCT AGT GTT SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 184. ATC TGT TCC ACA GTC ACA CGG GTT CC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 185. ATC TCT GGT TCC ACG ATG AGT TGT GTT CC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 186. ATC TGG CTC CAC GAA GAG TTT GAT GCC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 187. ATC TCT TAC GTT GTT GTA CCT CCA GAT AGG TTC C SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 188. ATC TGT CTT ACC TAC AAC TGT GAG TCT GGT GCC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 189. ATC TCC TTA CCT ACA ACG GTT AAC CTG GTC CC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 190. ATC TCT TAC TCA CCT ACA ACA GTG AGC CAA CTT CC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 191. ATC TAT ACC CAA GAC AGA GAG CTG GGT TCC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 192. ATC TAA CTT ACC TAG GAT GGA GAG TCG AGT CCC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 193. ATC TCT GTC ACA GTG AGC CTG GTC CC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 194. ATC TCA CGG TGA GCC GTG TCC C SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 195. ATC TCC AGT ACG GTC AGC CTA GAG CC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 196. ATC TCA CTG TCA GCC GGG TGC C SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 197. ATC TCA CTG AGA GCC GGG TCC C SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 198. ATC TAC CAG GAG CCG CGT GCC SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 199. ATC TCA CGG TCA GCC TGC TGC C SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 200. ATC TGA CCG TGA GCC TGG TGC C SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 201. ATC TGT GAA GTT ACT ATG AGC TTA GTC CCT TCA GCA AA SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 202. ATC TCG AAG TTA CTA TGA GCC TAG TCC CTT TTG CAA A SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 203. ATC TTG ACA ACA AGT GTT GTT CCA CTG CCA AA SEQ IDAAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCGNO: 204. ATC TCT GTA ATG ATA AGC TTT GTT CCG GGA CCA AA

In some embodiments, the composition is a first primer extension primermixture comprising V-gene specific primers for one or more immunesequence genes. In some embodiments, a first primer extension primermixture includes primers for one or more of TCR-alpha, TCR-beta,TCR-gamma, TCR-delta and immunoglobulin (IGH, immunoglobulin heavychain, IGL, immunoglobulin light chain-lambda and IGK, immunoglobulinlight chain-kappa). In some embodiments, the mixture comprises multipleprimers, e.g., 69 TCR-beta, 8 TCR-deta and 138 IGH V-gene primers.

In some embodiments, the composition is a second primer extension primermixture comprising J-gene specific primers for one or more immunesequence genes. In some embodiments, a second primer extension primermixture includes primers for one or more of TCR-alpha, TCR-beta,TCR-gamma, TCR-delta and immunoglobulin (IGH, immunoglobulin heavychain, IGL, immunoglobulin light chain-lambda and IGK, immunoglobulinlight chain-kappa). In some embodiments, the mixture comprises multipleprimers, e.g., 14 TCR-beta, 4 TCR-deta and 9 IGH J-gene primers.

In some embodiments, the V-gene primers and J-gene primers for variousgenes were selected from Table 2a.

TABLE 2a V-gene and J-gene primers primers SEQ ID NO: NAME SEQUENCEIGHV3-38 GGTAGCACATACTACGCAGACTCC IGHV3-22 GGACAACAGAATAGACCACGTCTGTIGHV3OR16-8 ACAACGCCAATAACTCACCGTATCT IGHVII-30-21GGAGCACAAATTACAACCCACTTCTC TRBV7-3 GGGCTGCCCAAAGATCGGT IGHV3-30CAGAGACAATTCCAAGAACACGCTG IGHV7-34-1 TGTCAGCACGGCGTGTCTT TRBV5-3AGGGCGCCAGTTCCATGAC TRBV15 ACACCCCTGATAACTTCCAATCCAG IGHV1-58CAGTGGTAACACAAACTACGCACAG TRBV12-1 CCTGATGTATCATTCTCCACTCTGAGGIGHV3OR16-6 GATTCACTTGCAGTAACGCCTGG IGHV1OR16-1GAGGGATCTACTCTGGCAATGGTAAG TRBV12-3 CAACAACGTTCCGATAGATGATTCAGG TRBV30TACTCCGTTGGTATTGGCCAGATC TRBV7-4 CAGAGTGATGCTCAACGAGACAAATC IGHV7-56TCTGTCAGCACGGCGGATC TRBV5-6 TCACCAGTTCCCTAACTATAGCTCTGA TRBV11-2CAGTTGCCTAAGGATCGATTTTCTGC TRBV1 CTGACAGCTCTCGCTTATACCTTCAT TRBV28CTCTAGAGAGAAGAAGGAGCGCTTC TRBV27 TGAAGGGTACAAAGTCTCTCGAAAAGA IGHVIII-82GCAACAGTGAGTTATCAGGGTTACTCT IGHVII-28-1 AATTGTATCTGCTTGCCCCTAGAAGATIGHV1OR15-3 CGCTGGCAATGGTAACACAAAATATTC IGHV1-24GTGAAACAATCTACGCACAGAAGTTCC IGHV7-4-1 CACCTCTGTCAGCACGGCA IGHV3-35GTAGGACGCACTATGCAGACTCTG TRBV6-5 CCACAGAGGATTTCCCGCTCA IGHV3-60GTGGTGATACCGTACTCTACACAGAC IGHV4-4 CCCTCAAGAGTCGAGTCACCATATC IGHVIII-5-2GGGGACAACACTTAACATCACAATCTC IGHV1OR16-4 GTGTCCTCCCCACAGGTGC TRBV10-1TTCACGACACTAACAAAGGAGAAGTCT IGHV1OR16-2 GTAAGACAGGCTATGCACAGAAGTTTCIGHV3-43 GTAGCACATACTATGCAGACTCTGTGA TRBV5-1 TCGATTCTCAGGGCGCCAG TRBV4-1CCAAGTCGCTTCTCACCTGAATG IGHV3-47 CCATCTCCAGAGACAACGCCA TRBV3-1AGTTCCAAATCGCTTCTCACCTAAATC IGHV1-2 TGGCACAAACTATGCACAGAAGTTTC IGHV3-54ATGTTATGCACAATCTGTGAAGAGCAG TRBV16 CAAGGAAAGATTTTCAGCTAAGTGCCT TRBV9AACATTCTTGAACGATTCTCCGCAC IGHV1-67 CCTGCAAGACTTGTGGATACACCTA IGHV7-27AGTCAAGTGAGACTTCACGCACT IGHV3-79 CGAGCAGATTCACCGTCTCCA IGHV3-63AGGACGTTTGTGTATTTTCAGGTGTTC TRBV24OR9-2 CGGTTGATCTATTGCTCCTTTGATGTCIGHVIII-11-1 TTGTACAGCCCAGCGGTTCA TRBV7-1 AGCAGACAAATCGGGGCTTCCIGHVII-53-1 CTCCAGATCCATGTCCAAAAAGCAG TRBVB CTGAGTCAGCATAGTCATTCCAACCAIGHV3OR16-10 AGAGACAATGCCAAGAACTCCTTGTAT TRBV13GATAAAGGAAGCATCCCTGATCGATTC IGHV1-17 AATGATAACACACACTACGCACAGAAG TRBV6-1CAATGTCTCCAGATTAAACAAACGGGA TRBV10-2 AGTCCCCGATGGCTACGTTG TRBV23-1CACAAGAAGCGATTCTCATCTCAATGC IGHV2OR16-5 AATGACAAAAAATCCTACAGCACGTCTIGHV3OR16-12 GTAGTAGTGGTTGTAGCACAAACTACG IGHV1-45GTAACACCAACTACGCACAGAAATTCC IGHV3-49 TACGCCGCGTCTGTGAAAGG IGHVII-43-1CTCCAGATCTATGTCCAAAAACAGCTC IGHV3-76 GGTACAGCTTGGGGGGTCC IGHVII-51-2GCATAGGTCACGAGGGAGCA IGHV5-51 AGTCCATCAGCACCGCCTAC IGHVIII-26-1TGGTTTCGGGTTTACTGGGTGC TRBV6-7 AATGTCTCCAGATCAAACACAGAGGAT TRBV7-6GGCTGCCCAATGATCGGTTC IGHV3-42 CTCATTTGCAGCTTCTAGATTCACCTT TRBV25OR9-2CTCCAGAATAAGGATAGAGCGTTTTCC TRBV8-2 GAACAGTGTTCTGATATCGACAAGACC TRBV5-2GGAAACTTGCCTAATTGATTCTCAGCT IGHVII-44-2 TCCTTCTCCAGAGATTCATCCAAGAAATRBV25-1 GTCTCCAGAATAAGGACGGAGCATTT IGHV8-51-1ATATGGCGTGGTGAAAGTCATCAATAC TRBV7-2 GGCTGCCCAGTGATCGCTT TRBV22-1GAGATCTAACTGAAGGCTACGTGTCT IGHV1OR15-6 TTGAGTGGATGGAACGTGTTGATCCIGHV1-68 TGGTAACACCAACTATGCAAAGAAGTT IGHVII-62-1CATAGGTCATGAAGGGAGCACACATTA IGHV3OR16-16 GTGACAGAACAGTGGCTATGTGTGIGHV3-50 CCTGAGACTCTGCCGTGCA IGHV7-81 CAGGGCTTCACAGGACGGT IGHV3-41CTCCAGAGACAATTCTAAGAGCATGCT IGHV3-29 GCCGAGTTCACCAGTCTCCA TRBV20-1CCACATACGAGCAAGGCGTC TRBV18 GCCAAAGGAACGATTTTCTGCTGAAT IGHV3-25GCAAAGCCTGCGTGGTCC IGHV3-15 CGCTGCACCCGTGAAAGG TRBV17CAGTACCAAAACATTGCAGTTGATTCA IGHVII-74-1 ACTACATCACAAACAGTGCTTATGACTTRBV19 AGAAAGGAGATATAGCTGAAGGGTACA IGHV7-40 ACCAACGGCTTCACAGGACGIGHVII-22-1 TGCTTCTCCATTACAACCAGTGCTT TRBV24-1AAAGGAGAGATCTCTGATGGATACAGT IGHV3-16 CTCCGTGAAGCGCCGATTC TRBV5-4TCTCCAGTTCCCTAATTATAGCTCTGA IGHVII-33-1 CATCTCCAGATCCATGTCCAAAAAGTAIGHV4-80 GGTGCAGCTGCAGGAGTGG IGHV1OR15-4 GGTAACACAACATATGCACAGAAGTTCTRBV14 CAACAATCGATTCTTAGCTGAAAGGAC TRBV5-5 AGCTCGCCAGTTCCCTAACTATAGIGHV3OR16-11 CACATACTATGCAGACTCCGTGAAG IGHV3OR15-7AGCTAACAGTTACACGACAGAATATGC IGHVIII-13-1 GTCAGACAGAGAAATACTACAGACCAGIGHVIII-38-1 ACTCGCCTTCAGTACAAAGAAGATTAA IGHV3-57AAGTGGGAGTTCTCAGAGTTACTCTCC IGHV1-69-2 GTCACCATAACCGCGGACAC IGHV1-14AGGGACACGTCCACGAGC TRBV29-1 CCAAACCTAACATTCTCAACTCTGACT IGHVII-65-1CATCCATCACCCCCCGCA IGHV3-32 GATGATGGAAGTCAGATACACCATGCA IGHV3-6GGGTCCCAGTTATTAGTGGTAGTGGTA IGHV3-52 AAAGTGTGACGGAAGTGAGAAATACTAIGHVIII-2-1 GAGTGATCAAGTATGAATTCTCAGGGT TRBV12-2CCTGATGGATCATTCTCTACTCTGAAG IGHV3-37 GAATGGGTCTCATACATTAGTGCTAGTTRBV20OR9-2 GGCTCCGAGGTCACATACGA IGHV4-55 TCGAATCACCATGTCCGTAGACAIGHVII-49-1 TGCTTCTCCATCACAACCAGTG TRBV6-8 GCTGCTGGTACTACTGACAAAGAAGTCTRBV7-5 CCTTCCAGGATGAAACTCAACAAGATA IGHV1-69D CACGATTACCGCGGACGAATTRBV26OR9-2 GAGATGTCTCTGAGAGGTATCATGTTT IGHV3OR16-7 CTGGAGTGGGTTGGCCGTATIGHV2-5 ATTTATTGGGATGATGATAAGCGCTAC TRBV21-1 AGAAAGCAGAAATAATCAATGAGCGATTRBV2 GCCTGATGGATCAAATTTCACTCTGA IGHV3-30-2 ACAGTGTGATGGAAGTCAGATATGTTATRBV22OR9-2 CTGAGACTGATCTATTACTCAAGGGTT TRBV26CCCTGTCTCTATTTGATCATCCATTTT TRBVA GCCGACTCATTATTCAGTTAACATTGA TRBV6-2GATGGCTACAATGTCTCCAGATTAAAA TRBV6-4 CCATTATTCAAATACTGCAGGTACCAC TRBV7-7CAATTATGAAGCTCAACCAGACAAATC TRBV7-9 CTTACTTCCAGAATGAAGCTCAACTAG TRBV10-3TGGTGTTAAAGATACTGACAAAGGAGA IGHV1OR15-2 ACACTTACAATGGTAACACAAACTACCIGHV3-36 TCATTTATGAGTTGTTGTGTAGGTAGC IGHV3-75GTCTCATGTATTAGTACTGATGGGAGT IGHVII-1-1 GCCCTCTGGGAAGGCGCT IGHVII-15-1GGATTTCCAATCATAACCAGTACTTCC IGHVIII-44 ACACTTTACAGACACCATCAATTTTCCIGHVIII-76-1 ACCCTCCATCAATACAAAGAAAAATCA IGHVIV-44-1GTGATATGGGTTAAGGGAAACACTAAG IGHV1-18 CGCTTACAATGGTAACACAAACTATGCIGHV2-70 TGGGATGATGATAAATACTACAGCACA IGHV2-70DTTGGGATGATGATAAATTCTACAGCAC IGHV4-28 CTATTATAGTGGGAGCACCTACTACAA IGHV6-1GTCCAAGTGGTATAATGATTATGCAGT IGHVII-26-2 TAGACTGGATCATCAAGGAATACACATIGHV3OR16-13 GCACAAGCTACGCAGACTCC IGHV3-13 GGTGACCCATACTATCCAGGCTTRBV8-1 AGCATGACCAAAGGCGGTG TRBV12-5 GCAACCGGGCTCCTCTAGA IGHV3-73ATGCTGCGTCGGTGAAAGG IGHV5-78 CCTTCCAAGGCCACGTCA IGHV1OR15-9AACCAGGGACACATCCATGG IGHV1OR21-1 TAGTGATGGCAGCACAAGCT IGHV1-69CACGATTACCGCGGACAAATC IGHV3OR16-9 GTGGTTACACAAACTACGCAGAC IGHV3OR16-15CGGTAAGACGCACTATGTGGA IGHV2-26 TCGAATGACGAAAAATCCTACAGC IGHJ3GTCACCGTCTCTTCAGGTAAGATGG TRBJ1-6 CTCTTGACTCGGGGGTGCC TRBJ2-4GCTCGGGTTTTTGTGCGGG TRBJ1-3 TGTTGTAGGTGAGTAAGTCAAGGCTG IGHJ2GAGTCCCACTGCAGCCCC IGHJ6 CCTCAGGTAAGAATGGCCACTCTAG TRBJ2-3CGTCGCAGGGCCAGTTTCT TRBJ2-7 AGTCGGAGGGTGGACCGG TRBJ1-5CTCTCCATCCTAGGTAAGTTGCAGAAT IGHJ1 GGTCACCGTCTCCTCAGGTG TRBJ2-6GTGAGTTTTCGCGGGACCAC IGHJ3P TACGTGGGAGGCCAGCAGA TRBJ1-2GTTAACCGTTGTAGGTAAGGCTGG TRBJ2-1 CACGACCCCAGAACCCTGT TRBJ1-4GATAGTGTATCATAAGGTCGGAGTTCC TRBJ2-2 TGGGTAAGGAGGCGGTTGG TRBJ2-5TTGGGTCTGGTTTTTGCGGG TRBJ1-1 TGTCCCTTTTAGAGTGGCTATATTCTT IGHJ2PGGTCTCAGCCCGGGGGTC IGHJ1P CCTACCAGCCGCAGGGTT TRBJ2-2PGCACCGGTTTTTGTCCTGG

In some embodiments, the composition is a second primer extensionmixture comprising J-gene specific primers and further comprising anopposite-facing primer capable of hybridizing to a primer binding site(universal primer binding site) in the first primer extension primer. Insome embodiments, the opposite-facing primer is a shortened sequencingprimer. In some embodiments, opposite-facing primer is a sequencingprimer to be used in a subsequencing sequencing step, the primer beingshortened by removing the index sequence.

Described herein are target polynucleotides and compositions containingsuch target polynucleotides. In some cases, a composition containingsuch target polynucleotides further contains a DNA-dependent polymeraseand/or a RNA-dependent DNA polymerase, a ligase, a first primer orplurality of first primers, a second primer or plurality of secondprimers, first or second primer polymerase extension products, or acombination thereof. Generally, the target polynucleotides encode immunecell receptors, such as B cell receptors (i.e., antibodies), or T cellreceptors, a complement thereof, or portions thereof. The targetpolynucleotides can be single stranded or double-stranded. The targetpolynucleotides can be DNA (e.g., genomic DNA). The targetpolynucleotides can be RNA (e.g., mRNA). The target polynucleotides canbe cDNA generated by reverse transcription of mRNA. In an exemplaryembodiment, the target polynucleotides are genomic DNA or cDNA that isheat denatured to form single-stranded targets.

In some embodiments, the target polynucleotides are obtained from asample enriched for immune cells. For example, the targetpolynucleotides can be obtained from a sample enriched for T cells,enriched for B cells, enriched for T cells and B cells, enriched forlymphocytes, or enriched for peripheral blood mononuclear cells (PBMCs).In some cases, the target polynucleotides are obtained from a sampleenriched for a fraction of T cells or B cells. For example, the samplecan be enriched for T cells that express α/β TCRs. As another example,the sample can be enriched for T cells that express γ/δ TCRs. As yetanother example, the sample can be enriched for B cells that express acertain isotype of BCR, or a set of such isotypes, such as IgA, IgG,IgM, IgE, or a combination thereof. As yet another example, the samplecan be enriched for B cells expressing kappa light chain BCRs. As yetanother example, the sample can be enriched for B cells expressinglambda light chain BCRs. As yet another example, the sample can beenriched for T cells that express α/β TCRs or γ/δ TCRs, wherein thesample is obtained by flow cytometry. As yet another example, the samplecan be a FFPE tissue sample containing infiltrating lymphocytes. As yetanother example, the sample can be a FFPE tissue sample, wherein thesample contains, or is suspected of containing, one or more tumor cells.Methods for enriching sample for a specific immune cell type include,but are not limited to, methods employing one or more of the following:ultracentrifugation, FICOLL™ gradient centrifugation, or flow cytometry(e.g., fluorescence-activated cell sorting (FACS)).

Described herein are reaction mixtures that contain targetpolynucleotides (e.g., target polynucleotides encoding B or T cellreceptors, a complement thereof, or portions thereof), first primers,first primer extension products (e.g., single-stranded or hybridized toa target polynucleotide), second primers, second primer extensionproducts (e.g., single-stranded or hybridized to a targetpolynucleotide), adapters (e.g., double stranded adapters, such assplint adapters), or a combination thereof. In some cases, the reactionmixture further contains a DNA-dependent DNA polymerase and/or aRNA-dependent DNA polymerase and reagents for polymerase-mediated andtemplate-directed primer extension (e.g., divalent cations such asmagnesium cations, nucleotide triphosphates, buffers, salts, etc.). Insome cases, the DNA polymerase exhibits strand-displacing activity. Insome cases, the DNA polymerase exhibits exonuclease activity. In somecases, the DNA polymerase does not exhibit or does not exhibitsubstantial strand-displacing activity. In some cases, the DNApolymerase does not exhibit or does not exhibit substantial exonucleaseactivity. In some cases, the DNA polymerase is thermostable. In somecases, the reaction mixture contains DNA ligase. In some embodiments,the RNA-dependent DNA polymerase is a telomerase. In some embodimentsthe RNA-dependent DNA polymerase lacks 3′-5′ exonuclease activity. Insome embodiments, the RNA-dependent DNA polymerase is thermostable.

In some embodiments, the DNA polymerase has long-range capability. Insome embodiments, the DNA polymerase has proofreading capabilityincluding processing proofreading capability. In some embodiments, theDNA polymerase has high fidelity. In some embodiments, the DNApolymerase is a mixture including Taq polymerase and an engineeredarchaeal B-family polymerase. In some embodiments, the polymerase hashot-start capability. In some embodiments, the hot-start capability isconferred by a thermolabile polymerase-specific antibody.

In some embodiments, the reaction mixture comprises an exonuclease. Insome embodiments, the exonuclease is a single-strand exonuclease. Insome embodiments, the exonuclease is thermolabile. In some embodiments,the exonuclease is inactivated at or below 95° C. In some embodiments,the exonuclease is inactivated at 80° C.

In some embodiments, the reaction mixture contains a plurality ofstructurally different target polynucleotides, wherein individual targetpolynucleotides of the plurality each comprise immune cell receptor Vgene regions, optionally D gene regions, optionally C gene regions, andJ gene regions (e.g., target polynucleotides encoding immune cellreceptor VJ, VDJ, or VJ and VDJ regions); and a plurality of firstprimers having [FW] regions. In some embodiments, the reaction mixturecontains a plurality of structurally different target polynucleotides,wherein individual target polynucleotides of the plurality each compriseimmune cell receptor V gene regions, optionally D gene regions,optionally C gene regions, and J gene regions (e.g., immune cellreceptor VJ, VDJ, or VJ and VDJ regions); and a plurality of firstprimers having [J] regions or [C] regions.

In some embodiments, the first primers of the reaction mixture arehybridized to the target polynucleotides of the reaction mixture. Insome embodiments, the reaction mixture contains a plurality ofstructurally different target polynucleotides hybridized to a pluralityof first primer extension products (i.e., products of aDNA-polymerase-mediated and template-directed extension reaction). Insome embodiments, the reaction mixture contains a plurality ofstructurally different immune cell receptor encoding targetpolynucleotides hybridized to a plurality of first primer DNA polymeraseextension products.

In some embodiments, the reaction mixture contains a plurality ofsingle-stranded target polynucleotides, the individual targetpolynucleotides each comprising the following from regions from 5′ to3′: a sequencer-specific adapter sequence, optionally a multiplexidentifier (MID) barcode, a unique molecular identifier (UID) barcode,at least a portion of an immune cell receptor framework 3 region, animmune cell receptor CDR3 region, an optional immune cell receptordiversity (D) region, an optional immune cell receptor constant (C)region, and at least a portion of an immune cell receptor J region, orthe complements thereof. In some cases, such a plurality ofsingle-stranded target polynucleotides are first primer extensionproducts. In some cases, such a plurality of single-stranded targetpolynucleotides are first primer extension products ligated to anadapter comprising a universal primer binding site and optionally an MIDbarcode.

In some embodiments, the reaction mixture contains a plurality ofsingle-stranded target polynucleotides, the individual targetpolynucleotides each comprising the following from regions from 5′ to3′: a sequencer-specific adapter sequence, optionally a multiplexidentifier (MID) barcode, a unique molecular identifier (UID) barcode,at least a portion of an immune cell receptor J-gene region, an optionalD region, an optional immune cell receptor constant (C) region, animmune cell receptor CDR3 region, and at least a portion of an immunecell receptor framework 3 region, or the complements thereof. In somecases, such a plurality of single-stranded target polynucleotides arefirst primer extension products. In some cases, such a plurality ofsingle-stranded target polynucleotides are first primer extensionproducts ligated to an adapter comprising a universal primer bindingsite and optionally an MID barcode.

In some embodiments, a reaction mixture can contain one or more of theforegoing first primer extension products (e.g., adapter ligated firstprimer extension products) hybridized to a plurality of second primerextension products. In cases where the first primers hybridize to aframework region of an immune cell receptor encoding targetpolynucleotide, the second primers can be configured to hybridize to a Jgene region or C gene region of the first primer extension products. Incases, where first primers hybridize to a complement of a J region or Cregion of an immune cell receptor encoding target polynucleotide, thesecond primers can be configured to hybridize to a framework region ofthe first primer extension products.

Described herein are adapters for downstream amplification or sequencingapplications. Attachment of an adapter to one or both ends of a targetpolynucleotide, first primer extension product, or second primerextension product, can attach a UID barcode, an MID barcode, a universalprimer binding site or complement thereof, or a combination thereof.Adapters containing a universal primer binding site or complementthereof can be referred to as universal adapters. In some embodiments,adapters are attached by ligation. In some embodiments, adapters areattached by hybridizing a primer containing an adapter sequence (e.g.,an adapter sequence comprising or consisting of a universal primerbinding site or complement thereof) to a target polynucleotide, firstprimer extension product, or second primer extension product. In someembodiments, the primer containing an adapter sequence is a secondprimer that can be hybridized to a first primer extension product andextended with a polymerase as described herein. In some embodiments,first adapters are ligated to a first primer extension product andsecond adapters are attached by hybridization of a second primercontaining such an adapter to the first primer extension product andextending the second primer.

In some embodiments, one or more adapters are splint adapters. Splintadapters can be hybridized to a [SPLINT] region of a primer extensionproduct (e.g., a first primer extension product) and ligated to a[5′-Phos] of the first primer extension product. Splint adapters cancontain a double stranded region and a 5′ single-stranded overhangregion, wherein the 5′ single-stranded overhang region is complementaryto and hybridizes under hybridization conditions with the [SPLINT]region of the primer extension product (e.g., a first primer extensionproduct). The 5′ single-stranded overhang region can be at least 2nucleotides in length, at least 4 nucleotides in length, at least 6nucleotides in length, at least 8 nucleotides in length, from 2 to 10nucleotides in length, or from 2 to 8 nucleotides in length. In someembodiments, the 5′ single-stranded overhang region comprises orconsists of 6 consecutive nucleotides that are complementary to the[SPLINT] region of the first primer extension product. In someembodiments, the 5′ single-stranded overhang region comprises orconsists of the sequence AGA TCG.

Splint adapters can contain a barcode and a universal primer bindingsite as described herein. In some cases, the splint adapter contains aMID barcode. In some cases, the splint adapter contains an MID barcodeand a universal primer binding site. In some cases, the barcode isencoded in the double-stranded region of the splint adapter. In somecases, the universal primer binding site is encoded in thedouble-stranded region of the splint adapter. In some cases, the barcode(e.g., MID barcode) and the universal primer binding site is encoded inthe double-stranded region of the splint adapter. In some embodiments,with respect to the strand of the splint adapter that contains the 5′single-stranded overhang region, in some cases, the universal primerbinding site can be 3′ of the barcode (e.g., MID barcode).

In some embodiments, the reaction mixture contains universalamplification primers hybridizing to adapter sequences or universalprimer binding sites in first and second round primer extension primers.The universal amplification primers may comprise sequencingplatform-specific sequences. Based on the platform, the sequencingprimers may include barcode (index) sequences, e.g., sample indexsequences. One or both sequencing primers may comprise index sequences.

In some embodiments, prior to universal amplification with universalprimers, excess primers from the second round of primer extension isremoved by an exonuclease. In some embodiments, the exonuclease is addedto a reaction mixture comprising excess extension primers to be degradedand also comprising universal amplification primers to be retained. Insome embodiments, universal amplification primers comprise one or moremodifications conferring exonuclease resistance.

In some embodiments, the nuclease-protecting modification allows toadvantageously skip a purification step between the second primerextension and universal amplification thus saving time, resources andreducing the possibility of sample contamination.

In some embodiments, the exonuclease is a 3′-5′-exonuclease and themodification is selected from one or more nucleotides at the 3′-endmodified with one of the following: a phosphorothioate (PS) bond, a2′-O-methyl (2′OMe), a 2′-fluoride and Inverted ddT. In someembodiments, the modification is a nucleotide with a phosphorothioate(PS) bond, which substitutes a sulfur atom for a non-bridging oxygen inthe phosphate backbone of an oligonucleotide. In some embodiments, themodification is a nucleotide with 2′-O-methyl (2′OMe) modification ofthe ribose/deoxyribose yielding 5- to 10-fold reduced susceptibility toDNases. In some embodiments, the modification is a nucleotide with2′-fluoro modification. The fluorine-modified ribose/deoxyribose conferssome relative nuclease resistance. In some embodiments, the modificationis a 2′,3′ dideoxy-dT base (5′ Inverted ddT) at the 3′ end of anoligonucleotide protecting against some forms of enzymatic degradation.In some embodiments, the universal primer has more than one modifiednucleotide and more than one type of modified nucleotide.

II. Methods

Described herein are methods for performing flanking primer (FP-) primerextension target enrichment (PETE) of target polynucleotides from asample. In some embodiments, the target polynucleotides encode immunecell receptors, or portions thereof. In such embodiments, the methodscan be referred to as immuno-PETE. The methods described herein canutilize a plurality of immune cell receptor V gene specific firstprimers each comprising an [FW] region at a 3′ end for hybridizing to aframework (e.g., framework 1, framework 2, or framework 3) region of atarget polynucleotide encoding an immune cell receptor. In suchembodiments, the plurality of second primers each comprise a [J] regionto flank the region of interest. Alternatively, the methods can utilizea plurality of immune cell receptor J gene specific first primers eachcomprising a [J] region at a 3′ end for hybridizing to a J region of atarget polynucleotide encoding an immune cell receptor. In suchembodiments, the plurality of second primers each comprise a [FW] regionto such that the first and second primers flank the region of interestin the target polynucleotides. In another embodiment, the methods canutilize a plurality of immune cell receptor C gene specific firstprimers each comprising a C-segment region at a 3′ end for hybridizingto a C gene region of a target polynucleotide encoding an immune cellreceptor. In such embodiments, the plurality of second primers can eachcomprise a [V] gene region such that the first and second primers flankthe region of interest in the target polynucleotides.

In some embodiments, the method includes: a) providing a reactionmixture containing: i) a plurality of structurally different targetpolynucleotides as described herein, wherein the individual targetpolynucleotides encode immune cell receptor V gene regions, optionally Dgene regions, optionally C gene regions, and J gene regions; and, ii) aplurality of immune cell receptor V gene specific primers or C genespecific primers (i.e., first primers) as described herein, wherein theimmune cell receptor V gene specific primers are hybridized to the Vgene regions of the target polynucleotides or wherein the immune cellreceptor C gene specific primers are hybridized to the C gene regions ofthe target polynucleotides; b), extending the hybridized immune cellreceptor V gene specific primers or C gene specific primers with apolymerase to generate extended immune cell receptor V gene specificprimers or extended immune cell receptor C gene specific primers (i.e.,first primer extension products) and then removing un-extended immunecell receptor V or C gene specific primers, if present, therebygenerating extended immune cell receptor V gene specific primerscontaining at least a portion of the immune cell receptor V region,optionally the immune cell receptor D region, and at least a portion ofthe immune cell receptor J region or at least a portion of the immunecell receptor C region, optionally the immune cell receptor D region,and at least a portion of the immune cell receptor V region. In somecases, the removing un-extended immune cell receptor V or C genespecific primers includes contacting the un-extended immune cellreceptor V or C gene specific primers with a single-stranded DNAexonuclease enzyme and thereby digesting the un-extended immune cellreceptor V or C gene specific primers.

In some embodiments, the invention is a method comprising the followingsteps: DNA extraction, optional DNA quality assessment, first round ofprimer extension, thermoabile exonuclease treatment, purification,second round of primer extension and optional purification therebyforming a library of enriched nucleic acid sequences. Depending on thedownstream use of the enriched library, the method may further compriseone or more of the following: library amplification, purification,quality control assessment, pooling of libraries and sequencing of thelibraries or pools.

In some embodiments, the first primer extension step comprises two ormore repetitions of the annealing step. In some embodiments, theannealing step comprises a temperature profile including several (e.g.,2, 3 or more) progressively lower annealing temperatures. The inventorshave discovered that utilizing a step-wise annealing procedure increasesspecificity of the target enrichment method. The inventors have furtherdiscovered that utilizing repetitions of the step-wise annealingprocedure further increases specificity of the target enrichment method.Disclosed herein is an improved primer extension target enrichmentmethod wherein the annealing temperature profile comprises one or moreof a series of decreasing temperatures. In some embodiments, theannealing step comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 or more roundsof thermocycling through the series of two or more annealingtemperatures. In some embodiments, by way of example, the annealing stepcomprises 20 cycles wherein each cycle consists of an incubation at 60°C., at 57.5° C. and at 55° C.

In some embodiments, the first primer extension step comprises a roundof denaturation, two or more rounds of step-wise annealing, and a roundof extension.

In some embodiments, the first primer extension step utilizes thefollowing temperature profile: [initial denaturation]-[two or morerounds of the annealing temperature profile]-[extension]. In someembodiments, by way of example, the first primer extension step utilizesthe following temperature profile:

Stage Temp (° C.) Duration Cycles Initial Denaturation 95° C. 10 min 1Intermediate Temp 80° C. 0 sec Annealing (ramp at 0.2° C./sec) 60° C. 0sec Annealing high 60° C. 20 sec 20 Annealing mid 57.5° C. 20 secAnnealing low 55° C. 20 sec Extension 72° C. 2 min 1 End 4° C. ∞ 1

In some cases, the removing un-extended immune cell receptor V or C genespecific primers includes solid phase reversible immobilization ofsingle-stranded first primer extension products or double strandedpolynucleotides containing single-stranded target polynucleotidehybridized to single-stranded first primer extension product. In somecases, the removing un-extended immune cell receptor V or C genespecific primers includes resin (e.g., silica (e.g., silica bead)) ormembrane-based column or batch purification of double-stranded DNA fromsingle-stranded DNA or purification of a selected size of single ordouble stranded DNA from the reaction mixture. In some cases, theremoving includes removing single or double-stranded DNA, or acombination thereof, having a size of less than about 100 bases or basepairs. In some cases, the removing includes removing single ordouble-stranded DNA, or a combination thereof, having a size of morethan about 1,000 bases or base pairs. In some cases, the removingincludes purifying from the sample primer extension products ordouble-stranded DNA containing primer extension products, or acombination thereof, having a size of more than about 100 bases or basepairs and less than about 1,000 bases or base pairs. In some cases, theremoving further removes genomic DNA or denatured (e.g.,single-stranded) genomic DNA from the sample. In some embodiments, theremoving further removes denatured cDNA from the sample.

In some embodiments, the method includes: c) hybridizing a firstuniversal adapter (e.g., a splint adapter containing a universal primerbinding site) to a [SPLINT] adapter hybridization site of the extendedimmune cell receptor V or C gene specific primers; d) ligating thehybridized first universal adapter to the extended immune cell receptorV or C gene specific primers, and then removing un-ligated adapters, ifpresent. In some cases, the removing un-ligated adapters includes solidphase reversible immobilization of adapter-ligated single-stranded firstprimer extension products or double stranded polynucleotides containingsingle-stranded target polynucleotide hybridized to such adapter-ligatedsingle-stranded first primer extension product. In some cases, theremoving un-ligated adapters includes resin (e.g., silica (e.g., silicabead)) or membrane-based column or batch purification of double-strandedDNA from single-stranded DNA or purification of a selected size ofsingle or double stranded DNA from the reaction mixture. In some cases,the removing includes removing from a reaction mixture single ordouble-stranded DNA, or a combination thereof, having a size of lessthan about 100 bases or base pairs. In some cases, the removing includesremoving from a reaction mixture single or double-stranded DNA, or acombination thereof, having a size of more than about 1,000 bases orbase pairs. In some cases, the removing includes purifying from thesample adapter-ligated primer extension products or double-stranded DNAcontaining such adapter-ligated primer extension products, or acombination thereof, having a size of more than about 100 bases or basepairs and less than about 1,000 bases or base pairs.

In some embodiments, removing the un-extended immune cell receptor V orC gene specific primers includes contacting the sample with anexonuclease. In some embodiments, the exonuclease has a single-stranddegrading activity and lacks the double-strand degrading activity. Insome embodiments, the exonuclease is thermolabile. In some embodiments,the exonuclease is Exonuclease I.

In some embodiments, the nuclease-protecting modification of universalamplification primers used in the amplification step allows toadvantageously skip a purification step between the second primerextension and universal amplification thus saving time, resources andreducing the possibility of sample contamination.

In some embodiments, the method includes: e), hybridizing a plurality ofimmune cell receptor J gene specific primers (i.e., second primers) tothe J region portions of the extended immune cell receptor V genespecific primers (i.e., first primer extension products), wherein theimmune cell receptor J gene specific primers comprise a 3′ J genehybridizing region and a 5′ second universal adapter region orhybridizing a plurality of immune cell receptor V gene specific primers(i.e., second primers) to the V region portions of the extended immunecell receptor C gene specific primers (i.e., first primer extensionproducts), wherein the immune cell receptor V gene specific primerscomprise a 3′ V gene hybridizing region and a 5′ second universaladapter region; and, f) extending the hybridized immune cell receptor Jgene specific primers or extending the immune cell receptor V genespecific primers with a polymerase, thereby forming a plurality ofstructurally different double-stranded products comprising an extendedimmune cell receptor J gene specific primer or extended immune cellreceptor V gene specific primer (i.e., second primer extension product)hybridized to an (e.g., adapter-ligated) extended immune cell receptor Vgene specific primer or extended immune cell receptor C gene specificprimer, each double-stranded product comprising at least a portion ofthe immune cell receptor V region, optionally the immune cell receptor Dregion, and at least a portion of the immune cell receptor J regionflanked by a first and second universal adapter sequence or at least aportion of the immune cell receptor C region, optionally the immune cellreceptor D region, and at least a portion of the immune cell receptor Vregion flanked by a first and second universal adapter sequence.

In some embodiments, the e) hybridizing and f) extending can be repeatedmultiple times by heating to denature double-stranded products producein the extending of f) (e.g., double-stranded DNA products comprisingfirst primer extension products (e.g., adapter-ligated extended immunecell receptor V or C gene specific primer) hybridized to second primerextension products (e.g., extended immune cell receptor J or V genespecific primer)), cooling to hybridize un-extended second primers tofirst primer extension products (e.g., adapter-ligated first primerextension products), and extending hybridized primers. In some cases, e)and f) are repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ormore times. In some cases, e) and f) are repeated from 2 to 15 times,from 3 to 12 times, from 5 to 10 times, or from 5 to 15 times.

As described herein after extension of hybridized second primers, apolynucleotide containing at least a portion of a J-region, an optionalD region, an optional C region, and at least a portion of a V region,flanked by universal primer binding sites, or a complement thereof isprovided. This polynucleotide can be amplified by universal PCR with anamplification reaction mixture containing the polynucleotide, a forwarduniversal primer and a reverse universal primer.

In some embodiments, extension of the second primer is performed in thepresence of the opposite-facing primer hybridizing to the primer-bindingsite introduced into the primer extension product by the first primer.

In some embodiments, the second primer extension step comprises two ormore repetitions of the annealing step. In some embodiments, theannealing step comprises a temperature profile including several (e.g.,2, 3 or more) annealing temperatures. In some embodiments, the annealingtemperature profile comprises a series of decreasing temperatures. Insome embodiments, the annealing step comprises 2, 3, 4, 5, 6, 7, 8, 9,10, 20 or more rounds of thermocycling through the series of two or moreannealing temperatures. In some embodiments, by way of example, theannealing step comprises 20 cycles wherein each cycle consists of anincubation at 60° C., at 57.5° C. and at 55° C.

In some embodiments, the second primer extension step comprises a roundof initial denaturation, two or more rounds of: denaturation, step-wiseannealing and extension, and a round of final extension. In someembodiments, by way of example, the second primer extension stepcomprises 10 cycles.

In some embodiments, the second primer extension step utilizes thefollowing temperature profile: [initial denaturation]-[two or morerounds of: denaturation, annealing temperature profile,extension]-[final extension]. By way of example, the following thermalprofile may be used.

Stage Temp (° C.) Duration Cycles Initial Denaturation 95° C. 5 min 1Denature 95° C. 30 sec 10 Annealing high 60° C. 10 sec Annealing mid57.5° C. 10 sec Annealing high 55° C. 10 sec Extension 72° C. 45 secFinal Extension 72° C. 1 min 1 End 4° C. ∞ 1

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that manymodifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only and are not meant to be limiting in anyway. It is intended that the specification and examples be considered asexemplary only, with the true scope and spirit of the invention beingindicated by the following claims.

All publications, patents, patent applications or other documents citedherein are hereby incorporated by reference in their entirety for allpurposes to the same extent as if each individual publication, patent,patent application, or other document was individually indicated to beincorporated by reference for all purposes. Where a conflict existsbetween the instant application and a reference provided herein, theinstant application shall dominate.

In some embodiments, the method further comprises contamination removalor reducing the amount of contaminating sequences in the sequencing dataoutput. As disclosed herein, the contamination filtering process is anovel combination of physical steps and analytical steps. The inventorshave observed that during amplification, a library may becomecontaminated by a nucleic acid fragment also containing universalamplification primer binding sites. For example, a fragment from anotherlibrary or a pool of libraries may contaminate a reaction mixturedescribed herein. In one embodiment, the primer extension targetenrichment method disclosed herein includes a method of detecting andremoving from sequencing data output the sequencing data originatingfrom contaminating nucleic acids, the method comprising: 1) an increasednumber of primer extension cycles in the first round of primerextension; alone or in combination with 2) one or more rounds ofexponential amplification in the second round of primer extension. Theinventors have discovered that these extra rounds increase the ratio oftrue targets to contaminants in the sample subjected to sequencing andfacilitate detection and removing of the sequencing data originatingfrom the contaminants from the sequencing data output. The additionalrounds of extension and added pre-amplification steps will increase theproportion of UMI (unique molecular index) family sizes of true targetsin a mixture comprising true targets and contaminants. The proportionsof molecule counts (by UMI sequences) are maintained by PCR, andsequencing. Therefore at the completion of sequencing, the contaminantreads appear as a much smaller population within the sequencing dataoutput. In some embodiments, a bi-modal UMI family size distribution isobtained (FIG. 11). For example, 8 rounds of amplification lead to anobserved 16-fold difference between the contaminant and an a target(FIG. 12: 64 reads vs. 1024 reads).

In some embodiments, the method further comprises after sequencing, astep of calculating the number of nucleic acid sequences belonging to aUMI family (a group of nucleic acid sequences sharing an identical UMI).In some embodiments, the method further comprises identifying andretaining in the sequencing data output the top 90% of UMI families bysize. In some embodiments, the method further comprises identifying andretaining in the sequencing data output the top 91%, 92%, 93%, 94%, 95%96%, 97%, 98% or 99% of UMI families by size. In some embodiments, themethod further comprises removing from the sequencing data output thebottom 10% of UMI families by size. In some embodiments, the methodfurther comprises removing from the sequencing data output the bottom9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of UMI families by size.

In some embodiments, the number of primer extension cycles in the firstround of primer extension and the number of primer extension cycles inthe second round of primer extension may be selected to optimize thecalculation described above. The inventors have found that selectingoptimal proportions of first round and second round primer extension maydecrease the amount of the output that needs to be removed in theaforementioned calculation step. In some embodiments, 10 primerextension cycles may be performed in the first round and 20 primerextension cycles may be performed in the second round in order tominimize the output data that needs to be removed when calculating thenumber of nucleic acid sequences belonging to a UMI family. For example,when 10 first round primer extension cycles and 20 second round primerextension cycles are performed, the method may require removing lessthan the bottom 10% of UMI families by size.

EXAMPLES Example 1: Experimental Protocol—Immuno-PETE Using gDNA fromHuman PMBC

An schematic of an exemplary embodiment of immuno-PETE is illustrated inFIG. 1.

A pool of primers selected from SEQ ID NOS:1-121 (i.e., first primerpool (gPE primer mix)) and a pool of the primers selected from SEQ IDNOS: 122-204 (i.e., second primer pool (PE2 primer mix)) were generated.The concentration of primers in each primer pool was adjusted to 100 μM,and the pools were diluted to a working concentration of 12 μM. As acontrol, a target enrichment method using a previously tested panel ofoncology related nucleic acid probes was analyzed in a separatereaction. Input human PMBC genomic DNA was used at 100 ng per reaction.Reaction mixtures were prepared and incubated as depicted below, inTable 3.

TABLE 3 Reagent Vol (μl) Primer Hybridization Mix Isothermal Amp Buffer(10x) 4 dNTPs (10 mM) 1 gPE primer mix (100 uM) 2 MgCl2 (25 mM) 6 total13 Aliquot 13 μl of Primer Hybridization Mix into each reaction tubesAdd water and samples to reaction tubes and mix as indicated above inthe table Place in thermocycler and run program Hyb and Extend 7-28Polymerase Master Mix Water 8 Isothermal Amp Buffer (10x) 1 NEB Bst 2.0(8 U/ul) 1 total 10 At 60° C. hold step, add 10 μl Polymerase Master Mixin each tubes and mix Go to run, edit, skip step and contunue with runprogram Hyb and Extend 7-28 Add Exonuclease 1 to samples 1 μl Runprogram Exo 1 7-28 Perform AMPure clean up at 1.4 ratio KAPAPure to add70 Elution Volume 20 Ligation Master Mix Water 3.9 T7 Ligase Buffer (2x)25 T7 DNA ligase (3000 U/μl) 0.1 total 29 Aliquot 29 μl Ligation MasterMix in each reaction tube Add 20 μl gPE Extension Product and Mix Add 1μl of C7/P7 BC (Barcode) adapter (25 uM) Incubate at 25° C. for 5minutes Add 50 μl of water to reach a volume of 100 μl Perform AMPureclean up at 0.7X ratio KAPAPure to add 70 Elution Volume 18 PrimerExtension 2 (PE2) Master Mix Phusion Mix (2X) 25 PE2 primer mix (20 uM)2 Ammonium sulfate (200 mM) 5 total 32 Aliquot 32 μl PE2 Master Mix ineach reaction tube Add 18 μl Ligation Product and Mix Place inthermocycler and run program PE2 for 15 Cycles Perform AMPure clean upat 1X ratio KAPAPure to add 50 Elution Volume 20 PETE CR Master Mix - 50uL Water 4 Phusion Mix (2X) 25 C5/C7 primer Mix (10 uM) 1 total 30 Add20 μl of the Primer extension 2 product Place in thermocycler and runprogram PETE CR Perform AMPure clean up at 0.9X ratio KAPAPure to add 45Elution Volume 20

Thermal Cycling Protocols are depicted in Table 4.

TABLE 4 I. Hyb and Extend 7-28 Program II. 98° C. - 2 minutes III. 80°C. - 0 seconds IV. 60° C.- 20 minutes V. 60° C. - hold VI. 65° C. - 2minutes VII. 80° C. - 10 minutes VIII. 4° C. - hold IX. Exo I 7-28Program X. 37° C. - 10 minutes XI. 80° C. - 10 minutes XII. 4° C. - holdXIII. PE2 Program XIV. 98° C. - 1 minute i. 15 cycles XV. 98° C. - 10seconds XVI. 80° C. - 20 seconds XVII. 64° C.- 20 seconds XVIII. 72°C. - 30 seconds XIX. 4° C. - hold XX. PETE CR Program XXI. 98° C. - 1minute XXII. 24 cycles XXIII. 98° C. - 10 seconds XXIV. 60° C. - 20seconds XXV. 72° C. - 30 seconds XXVI. 4° C. - hold

Immuno-PETE reactions were analyzed by agarose gel electrophoresis (FIG.2). The TCR library (generated with first primers (selected from SEQ IDNOS: 1-121) and second primers (selected from SEQ ID NOS:122-204) andRH-4 library (generated with the oncology panel of probes (Control))were diluted to a final concentration of 4 nM, and pooled in a 4:1 ratioof TCR to RH-4 for Illumina sequencing. Sequencing was performed on theIllumina MiSeq instrument using paired-end 2×250 cycle sequencing. Over15 million filtered pass reads were generated and analyzed.

Results:

The results of the TCR library and RH-4 library are summarized in Table5.

TABLE 5 Sequencing Summary Reads Mapped to Index ID % Reads Total PFIndentified Reads Reads (PF) CV Min Max 15805692 15271036 85.4589 0.827717.7223 67.7366 Index Sample Index Index % Reads Number Is Project 1(i7)2(i5) Indentified 1 TCR-PETE NA ATCACG 67.7366 2 RH4-PETE NA TGACCA17.7223

A random subsample of 150,000 sequences, corresponding to 50× theexpected number of different V(D)J sequences in the input genomicsample, was further analyzed. This subsample of sequencing data wasde-duplicated. The de-duplicated (12K) and de-deduplicated (150K) datawere processed to identify polynucleotides containing rearranged V(D)Jregions. The results are summarized in Table 6.

TABLE 6 TCR Alignment Result Total % read input Total input pairs readsUnique Duplicate_ read after after read read_ Sample pairs filteringfilter pair pairs TCR-PETE_1_sampled 150000 139654 93.1 2376 127231 50×TCR-PETE_S1_  12000  11178 93.2 1418  8955 sampled_4× TCRB Un-productive assigned Con- unique read_ %_Dup sensus consensus Shannon_Sample pairs Rate reads reads Clones d50 entropy TCR 10018 98.2 1699 174108 0.96296296 100.8085935 PETE_l_sampled 50× TCR-  803 86.3 1081 105 99 0.97979798  98.0031575 PETE_S1_ sampled_4×

Out of the 1081 sequences assigned in the 12k sample to 99 clones, 100%have the same clone assigned when they were present in the 150k set.There were in total 108 clones assigned to both sets, with 99 clonesoverlapping between the two sets. A Venn diagram of the overlap betweenthe 12k set and the 150k set is depicted in FIG. 3.

An analysis of top ranked TCR clones from the sample before and afterde-duplication showed that the ranking can change as a result ofde-duplication, as shown in Table 7.

TABLE 7 Top 10 ranking clones for TCRαβ and TCRγδ isotypes (TRA -Top10-Non-Dedup SEQ ID NO: 214-223; TRA -Top10-Dedup: SEQ ID NO 244-253; TRB-Top10 - Non-Dedup: SEQ ID NO 224-233; TRB Top10 - Dedup: SEQ ID NO:254-263; TRG - Top10 - Non-Dedup: SEQ ID NO: 234-243; TRG -Top10- Dedup:264-273) Clone Rank Clone TRA - Top10 - Non-Dedup 1TRAV27*_TRAJ42*_CAGGGSQGNLIF 2 TRAV12-2*_TRAJ43*_WPTGMRF 3TRAV38-1*_TRAJ33*_CAFMTPDSNYQLIW 4 TRAV21*_TRAJ45*_CAVGGYSGGGADGLTF 5TRAV25*_TRAJ28*_CAGSGAGSYQLTF 6 TRAV1-2*_TRAJ27*_CAVSTNAGKSTF 7TRAV13-1*_TRAJ18*_CAAKGRGSTLGRLYF 8 TRAV27*_TRAJ42*_CAGGGSQGNLIF 9TRAV26-1*_TRAJ47*_CIVRVVEYGNKLVF 10 TRAV5*_TRAJ4*_CAESEDSGGYNKLIF TRA -Top10 - Dedup 1 TRAV27*_TRAJ42*_CAGGGSQGNLIF 2 TRAV12-2*_TRAJ43*_WPTGMRF3 TRAV38-1*_TRAJ33*_CAFMTPDSNYQLIW 4 TRAV21*_TRAJ45*_CAVGGYSGGGADGLTF 5TRAV25*_TRAJ28*_CAGSGAGSYQLTF 6 TRAV13-1*_TRAJ18*_CAAKGRGSTLGRLYF 7TRAV27*_TRAJ42*_CAGGGSQGNLIF 8 TRAV1-2*_TRAJ27*_CAVSTNAGKSTF 9TRAV26-1*_TRAJ47*_CIVRVVEYGNKLVF 10 TRAV13-2*_TRAJ13*_CAEMAPGGYQKVTFTRB - Top10 - Non-Dedup 1 TRBV19*_TRBJ2-7*_CASSIRSSYEQYF 2 TRBV4-3*,TRBV4-2*_TRBJ2-1*_CASSLLSYNEQFF 3 TRBV20-1*_TRBJ2-7*_CSAPRTSGGLLNPYEQYF4 TRBV7-9*_TRBJ2-7*_CASSSQEAGGRYNSYEQYF 5TRBV7-8*_TRBJ2-5*_CASSLKRDGQETQYF 6 TRBV5-1*_TRBJ2-7*_CASSLEGQASSYEQYF 7TRBV3-1*, TRBV3-2*_TRBJ2-3*_CASSLGTDTQYF 8TRBV7-2*_TRBJ2-2*_CASSLGVGTGELFF 9 TRBV7-9*_TRBJ2-7*_CASSSEIGTAGTSHNRQYF10 TRBV19*_TRBJ2-7*_CASSVRSSYEQYF TRB - Top10 - Dedup 1TRBV19*_TRBJ2-7*_CASSIRSSYEQYF 2 TRBV4-3*,TRBV4-2*_TRBJ2-1*_CASSLLSYNEQFF 3 TRBV20-1*_TRBJ2-7*_CSAPRTSGGLLNPYEQYF4 TRBV5-1*_TRBJ2-7*_CASSLEGQASSYEQYF 5 TRBV7-8*_TRBJ2-5*_CASSLKRDGQETQYF6 TRBV7-9*_TRBJ2-7*_CASSSQEAGGRYNSYEQYF 7TRBV7-2*_TRBJ2-2*_CASSLGVGTGELFF 8 TRBV3-1*,TRBV3-2*_TRBJ2-3*_CASSLGTDTQYF 9 TRBV28*_TRBJ2-7*_CASSLDRNEQYF 10TRBV19*_TRBJ2-7*_CASSVRSSYEQYF TRG - Top10 - Non-Dedup 1 TRGV9*_TRGJ2*,TRGJ1*_CALWEVPHYYKKLF 2 TRGV4*_TRGJ2*, TRGJ1*_CADQPQAYKKLF 3TRGV9*_TRGJP1*_CALWDETGWFKIF 4 TRGV11*_TRGJP1*_CACWIRHVRATGWFKIF 5TRGV5*, TRGV3*_TRGJ2*, TRGJ1*_CATWDRPEKLF 6 TRGV3*, TRGV5*_TRGJ2*,TRGJ1*_CATWDNPYYKKLF 7 TRGV3*, TRGV5*_TRGJ2*, TRGJ1*_CATWDSLYYKKLF 8TRGV9*_TRGJP1*_CALWEVLTLSRTTGWFKIF 9 TRGV10*_TRGJP1*_CAAHTTGWFKIF 10TRGV4*_TRGJ2*, TRGJ1*_CATCLYYKKLF TRG - Top10 - Dedup 1 TRGV9*_TRGJ2*,TRGJ1*_CALWEVPHYYKKLF 2 TRGV4*_TRGJ2*, TRGJ1*_CADQPQAYKKLF 3TRGV9*_TRGJP1*_CALWDETGWFKIF 4 TRGV11*_TRGJP1*_CACWIRHVRATGWFKIF 5TRGV5*, TRGV3*_TRGJ2*, TRGJ1*_CATWDRPEKLF 6 TRGV3*, TRGV5*_TRGJ2*,TRGJ1*_CATWDNPYYKKLF 7 TRGV3*, TRGV5*_TRGJ2*, TRGJ1*_CATWDSLYYKKLF 8TRGV4*_TRGJ2*, TRGJ1*_CATCLYYKKLF 9 TRGV9*_TRGJP1*_CALWEVLTLSRTTGWFKIF10 TRGV10*_TRGJP1*_CAAHTTGWFKIF

Example 2: Experimental Protocol—RNA or mRNA Based Immuno-PETE

A modification of the immuno-PETE assay (e.g., as set forth inExample 1) is provided for starting material comprising total RNA orpurified mRNA. Total RNA or purified mRNA can be obtained from wholeblood, PBMC, sorted lymphocytes, lymphocyte culture, fresh orfresh-frozen tumor tissue, FFPE tissue samples, and the like. Aschematic outlining an exemplary method for RNA or mRNA basedimmuno-PETE is set forth in FIG. 4. Briefly, a cDNA synthesis step isintroduced prior to the immuno-PETE method essentially set forth inExample 1.

Here, an oligo-dT primer, set of random primers (e.g., hexamers,heptamers, octamers or nanomers, etc.,) or one or more C-segment primers(e.g., comprising one or more C-segment primers selected from SEQ IDNOS:205-213; see Table 8) is added to an aliquot of total RNA orpurified mRNA to form a reaction mixture. A reverse transcriptase (e.g.,SuperScript III™) and amplification components (such as buffers, dNTPsand salts (e.g., MgCl₂)) is added to the reaction mixture to initiatefirst and second strand synthesis to form double-stranded cDNAmolecules. The cDNA molecules can be purified, for example using SPRIbeads, and quantified for later use. The purified cDNA is then used as astarting template for the Immuno-PETE protocol set forth in Example 1.For example, purified cDNA is used as a starting template and a V genespecific probe set (e.g., comprising SEQ ID NOS:1-121) is used in thefirst round of extension (gPE primer pool), followed by second round ofextension using the C-segment probe set (PE2 primer pool, e.g.,comprising SEQ ID NOS:205-213). Alternatively, the first round ofextension can include use of the C-segment gPE primers, followed by asecond round of extension using the plurality of V gene specific PE2primers (e.g., comprising SEQ ID NOS:122-204).

Several advantages are achieved through the application of a cDNAsynthesis step to the immuno-PETE assay (e.g., as described inExample 1) including (1) improved assay sensitivity (more copies ofimmune receptor mRNA exist in immune cells as compared to the singlecopy of somatically rearranged V(D)J locus in genomic DNA); (2) decreasein amplification bias (use of fewer C-segment primers/probes as comparedto J-segment primers/probes in the immuno-PETE protocol of Example 1);(3) identification of immune receptor isotypes (resulting amplions fromthe RNA or mRNA based Immuno-PETE assay span the V(D)J immune receptordomain. For TCR, there are α-1, β-1 and β-2 constant genes. Forimmunoglobulins, the distinguishable isotypes are κ and λ for lightchains, and IgA, IgD, IgG, and IgM for the heavy chains). As such, thetotal RNA and/or mRNA based immuno-PETE assay is advantageous forhigh-throughput sequencing and/or immune repertoire profiling.

TABLE 8 Sequence Sequence ID name Sequence 5′→3′ 205 IgMGATGGAGTCGGGAAGGAAGTCCTGTGCGAG 206 IgG GGGAAGACSGATGGGCCCTTGGTGG 207 IgACAGGCAKGCGAYGACCACGTTCCCATC 208 IgD CCACAGGGCTGTTATCCTTT 209 IgEAGGGAATGTTTTTGCAGCAG 210 Igκ CATCAGATGGCGGGAAGATGAAGACAGATGG TGC 211 IgλCCTCAGAGGAGGGTGGGAACAGAGTGAC 212 TCRB GCTCAAACACAGCGACCTCGGGTGGGAACAC213 TCRA TCTCTCAGCTGGTACACGGCAGGGTCAGGG

Example 3: Combined Immuno-PETE and Oncology Targets Panel

In immuno-oncology applications, it would be advantageous to profileboth the somatic mutations in a tumor cell genome and the clonotyperepertoire of tumor infiltrating T-cells, using a sample of tumor tissue(e.g., tumor biopsy or FFPE tumor tissue). Designing and optimizing suchan assay by repeated design of multiple opposing (forward and reverse)multiplex-PCR primers is not practical. However, Immuno-PETE minimizesprobe-probe (or primer-primer) interactions by using a design algorithmthat separately designs gPE probe sets and PE2 probe sets to minimizeprimer dimers. The design algorithm can also place primers on +/−strands of genomic DNA such that primers are not pointed toward eachother in the same reaction step, to prevent production of a shortertarget. A further feature of the immuno-PETE assay is that each of thetwo probe hybridization and extension steps of the assay occur inseparate (distinct) reactions after removal of residual probes from theprevious step. Here, we demonstrate the immuno-PETE assay efficientlytargets cancer genes and T-cell receptors in a combined assay, withoutprior optimization of the combined probe set, where there was no overlapor probe coordinates between the Oncology and TCR gene targets.

Experimental Design

In order to demonstrate the utility of enriching both tumor cell genometargets and the somatically rearranged TCR targets from immune cells, wecombined an oncology panel (Signature or “Sig” panel) containing 181target regions from 21 cancer genes with a panel of V (n=60) and J(n=13) gene probes targeting TCRB. Specifically, SEQ ID NOS:1-60 wereused as the V-gene probes and SEQ ID NOS:188-200 were used as the J-geneprobes. Each of the panels was tested independently, but no attemptswere made to optimize the combined probe set, all probes were used atequimolar concentrations.

Protocol:

Human genomic DNA (Promega Cat #G1471) was used as an input at 100 ng or450 ng per reaction. This DNA sample is a mixture from multiple donors;it is not expected to contain oncology relevant somatic mutations, andassuming it has been isolated from PBMC, it contains T-cell genomic DNA.TCRB repertoire is expected to be very diverse due to the pooled natureof the DNA sample. Reaction mixtures were prepared and incubated as setforth in Table 9.

Results:

After completion of the immuno-PETE protocol set forth in Table 9, analiquot of the reaction products from the 100 ng and 450 ng input gDNAwas analyzed by agarose gel electrophoresis (FIG. 5). In FIG. 5, lanes1-8 correspond as follows, lane 1: MW ladder; lanes 2,3: Oncology panelonly, 100 ng gDNA; lanes 5, 6: Oncology panel only, 450 ng gDNA; lane 4:Oncology panel+TCRB, 100 ng gDNA; lane 7: Oncology panel+TCRB, 450 nggDNA; lane 8: No Template (NT), negative control.

The enriched libraries were sequenced using Illumina MiSeq sequencer andIllumina MiSeq sequencing kit v3 using 2×300 cycles as permanufacturer's protocols.

Sequencing Analysis:

Analysis of oncology targets in the combined panel assay are provided inTable 10. A total of 543,000 read pairs for each of the 100 ng and 450ng input gDNA was assessed. For the 450 ng gDNA sample a total of153,138 unique pairs were identified. By comparison, 118,098 uniquepairs were identified in the 100 ng gDNA sample. Additionally, analysisof the TCRB targets in the combined panel assay for each of the 100 ngor 450 ng input gDNA are summarized in Table 11.

TABLE 10 PETE sw % Total % Mean bases Total read Deduped Deduped DedupDedup UIDs per >= input pairs trimmed trimmed On- % non- Median 100×Uniformity( read after UNIQUE reads reads target On- zero target Cover0.5×-2× of Sample pairs filtering pairs % Duplicate mapped mapped readstarget probe coverage age mean) 450 ng-Sig- 543000 520443 153138 68.2152217 29.2 138948 91.3 846.1 1367 99.9 81.2 TCRb_S6_ L00 100 ng- 543000522924 118098 75.2 117291 22.4 101989   87 652.5  970 99.6 77.9 Sig-TCRb_S5_ L00

TABLE 11 (SEQ ID NO: 318-337) Total_ TCRB_ Total_ reads_ %_input_ Dupli-Un- productive_ input_ pairs_ reads_ Unique_ cate_ assigned_ % unique_read_ after after_ read_ read_ read_ Duplicate_ Consensus_ consenus_Shannon_ Sample pairs filtering filtering pairs pairs pairs Rate readsreads d50 entropy 100 ng- Sig- TCRb_ S5_L001 3036337 2923802 96.3 7488265 2912059 91.7 747 85 0.97619048 83.6249492 Clone Clone countfraction AA. Seq. CDR3 2 0. CATSRGSPNYGYTF 02352941 1 0.CASSPGTSGSASSTDTQ 01176471 YF 1 0. CASSLALIVGGENTEAF 01176471 F 1 0.CASSKGGTGGGWAGE 01176471 LFF 1 0. CASSQAFLPSLVTDTQ 01176471 YF 1 0.CASSADTGTFMNTEAF 01176471 F 1 0. CASSLGERGALSETQY 01176471 F 1 0.CASSFSKN 01176471 SRPYNEQFF 1 0. CASSLGSRGQRLLEQY 01176471 F 1 0.CASSLVAGGFSYNEQF 01176471 F 450 ng Sig- TCRb_ S6_L001 1393309 133559995.9 580 2904 1330803 83.4 569 140 0.98550725 137.254685 Clone Clonecount fraction AA. Seq. CDR3 2 0. CASSPGTGIDTQYF 01428571 2 0.CASSRQGNSPLHF 01428571 1 0. CASSQGRTRLQRGGRT 00714286 DTQYF 1 0.CASSIGLAGALRDTGEL 00714286 FF 1 0. CASSLRGPGQGEGGSP 00714286 LHF 1 0.CASSYPFPLTGGNQPQ 00714286 HF 1 0. CASSYAGTRLGNQPQH 00714286 F 1 0.CASSLGLAGVRQETQY 00714286 F 1 0. CASSLNRAFSGANVLT 00714286 F 1 0.CASSEARSGPDTDTQY 00714286 F

Here, the data demonstrates the potential of immuno-PETE assays tosequence both oncology targets in the tumor cell genome and theclonotype repertoire of tumor infiltrating T-cells. The combined assayis a single tube reaction requiring as little as 100 ng DNA input, thusmaking it suitable for analysis of tumor tissue biopsies or FFPE tissuesamples in immuno-oncology applications.

Immune-sequencing assays compatible with clinically relevant sampletypes, such as enriched T cell population or formalin fixed paraffinembedded tissue (FFPE) would provide valuable clinical utility.

Example 4 demonstrates Immuno-PETE, as described herein, in the contextof FACS sorted T cells; while Example 5 demonstrates Immuno-PETE in thecontext of FFPE tissue cells.

Example 4: Immuno-PETE Using Antigen Positive FACS Sorted Human T-CellsExperimental Design

The amino acid sequences of CDR3 were determined for T cell receptoralpha and beta chains. Here, Immuno-PETE was performed essentially asset forth in Example 1 with DNA extracted from formalin fixed human Tcells that were FACS sorted for M1 antigen (using 113 ng, 57 ng, or 11ng DNA input; see Table 12 and FIG. 6).

Results:

Clonotyping of T cell receptor alpha and beta chains was determined, seeTable 12 and FIG. 6.

TABLE 12 Sample type ng T cell count * FACS sorted M1 specific T cells113 18,880 57 9,440 11 1,888 FFPE 90% Tumor 10% 27 443 M1 specific Tcells 6 100 * 1 TCR template per 6 pg of DNA

Conclusion

This example demonstrated the ability to predict T cell receptor alphaand beta CDR3 sequences at low input amounts in FACS sorted T cells. TheT cell population was highly clonal as expected, and the most frequentlyobserved CDR3 sequences were the same for all input amounts, for bothalpha and beta chains.

Here, the immuno-PETE assay utilized at most 113 ng DNA input and aslittle as 11 ng DNA input, thus making it very suitable for analysis oftumor tissue biopsies or FFPE tissue samples in immuno-oncologyapplications.

Example 5: Immuno-PETE Using FFPE Tissue Sample Experimental Design

As in Example 4, the amino acid sequences of CDR3 were determined for Tcell receptor alpha and beta chains. Here, human T cells that were FACSsorted for M1 antigen were mixed with adenocarcinomic human alveolarbasal epithelial cells (A549) at 1:9 ratio. Mixed cells were pelletedand fixed with formalin and embedded in paraffin (FFPE). DNA wasextracted from the sectioned FFPE cell pellet, and Immuno-PETE wasperformed essentially according to Example 1 with either 6 ng or 27 ngDNA input. The DNA input amount was determined with qPCR (KAPA HumanGenomic DNA Quantification and QC kit, Catalog Number 07960590001, RocheDiagnostics Corp., IN, USA).

Results:

Clonotyping of T cell receptor alpha and beta chains was determined, seeTable 12 and FIG. 7.

Conclusion

This Example demonstrates the ability to predict T cell receptor alphaand beta CDR3 sequences at low input amounts in FFPE tissue cells mixedwith tumor cells (1:9 ratio). The T cell population was highly clonal asexpected, and the most frequently observed CDR3 sequences were the samefor both input amounts, for both alpha and beta chains.

Here, the immuno-PETE assay utilized less than 30 ng DNA input making itvery suitable for analysis of tumor tissue biopsies or FFPE tissuesamples in immuno-oncology applications.

Example 5. Improved Method of Immuno-PETE

In this experiment, genomic DNA was isolated from PBMC, PanT cells ofHut78 cell line. The first Primer Extension reaction had primers for Vgenes of: TCR-beta (69), immunoglobulin (immunoglobulin heavy chain,IGH) (138) and TCR-delta (8), the reaction contained:

Reagent Volume (ul) Kapa Long Range HotStart Ready Mix (2X) with Dye12.5 V-gene primer mix (50 uM) 1 DMSO (to final volume in reaction of5%) 1.25 sample gDNA(100 ng) 10.25 Total 14.75The following thermocycling program was run:

Stage Temp (° C.) Duration Cycles Initial Denaturation 95° C. 10 min 1Intermediate Temp 80° C. 0 sec Annealing (ramp at 0.2° C./sec) 60° C. 0sec Annealing high 60° C. 20 sec 20 Annealing mid 57.5° C. 20 secAnnealing low 55° C. 20 sec Extension 72° C. 2 min 1 End 4° C. ∞ 1A 1:5 dilution of Thermoabile Exonuclease I in nuclease-free water wasmade and added at 5 ul per sample. The following thermocycling programwas run:

Temp (° C.) Duration 37° C. 4 min 80° C. 1 min  4° C. ∞The first Kapa HyperPure bead purification was performed according tothe manufacturer's instructions resulting in dry beads in each vial.The second primer extension reaction had primers for J genes of:TCR-beta (14), immunoglobulin (immunoglobulin heavy chain, IGH) (9) andTCR-delta (4). The reaction also contained an opposite facing universalprimer (Illumina i7 without barcode/index sequence). The reactioncontained:

Reagent V (ul) Kapa Long Range HotStart Ready Mix (2X) with Dye 12.5J-gene primer mix (20 nM) 2 i7-short (no index) primer 1 Nuclease-freewater 9.5 Add to dry beads from 1^(st) primer extension Total 25The following thermocycling program was run:

Stage Temp (° C.) Duration Cycles Initial Denaturation 95° C. 5 min 1Denature 95° C. 30 sec 10 Annealing high 60° C. 10 sec Annealing mid57.5° C. 10 sec Annealing high 55° C. 10 sec Extension 72° C. 45 secFinal Extension 72° C. 1 min 1 End 4° C. ∞ 1The reactions proceed directly to library amplification. The libraryamplification reaction contained:

Reagent V (ul) Kapa Long Range HotStart Ready Mix (2X) with Dye 12.5Nuclease-free water 2.5 Thermoable ExoI (1:5 diluted) 5 Add to 2^(nd)primer extension reaction 25 Nuclease-protected i7/i5 indexed sequencingprimers 5 Total 50The following thermocycling program was run:

Stage Temp (° C.) Duration Cycles Reaction 37° C. 4 min 1 InitialDenaturation 95° C. 3 min 1 Denature 95° C. 30 sec 20 Anneal 57° C. 30sec Extension 72° C. 45 sec Final Extension 72° C. 1 min 1 End  4° C. ∞1The first Kapa HyperPure bead purification was performed according tothe manufacturer's instructions. The quality and concentration ofresuspended libraries were analyzed on Bioanalyzer and Qubit Quant. Thefollowing quality control standards were set for Immuno PETE:1. The concentration of the libraries should be over 0.5 ng/ul2. No or very few small fragments detected under 200 bp3. No or few large fragments over 700 bp4. Narrow peak for cell linesThe QC-pass libraries were pooled and sequenced on Illumina NextSeqinstrument following manufacturer's instructions.

Example 6. Contamination Removal from Sequencing Data

In this example, contamination removal was applied to the sequencingreads obtained from a sample having nucleic acids isolated from PanTspiked with a contaminant from the Hut78 cell line. Primer extension wasperformed by the method of Example 5. The enriched nucleic acids weresequenced on Illumina NextSeq instrument.

The peak of 64 reads was observed corresponding to the contaminant.(FIG. 12). VDJ rearrangements and Clone Counts for all UMI familieswith >1 reads were obtained. Even after filtering UMI families sizesfor >1 reads (replicates), 966 clones of the Hut78 sequence wereobserved contaminating the PanT sample. After filtering for the top 90%of UMI families (covered by 90% of the total clustered reads) a majorityof the Hut78 clone sequences (UMI families) were filtered out and only38 remained. The remainder of the VDJ recombinants left in the samplewere sequences from the PanT sample.

While the invention has been described in detail with reference tospecific examples, it will be apparent to one skilled in the art thatvarious modifications can be made within the scope of this invention.Thus the scope of the invention should not be limited by the examplesdescribed herein, but by the claims presented below.

We claim:
 1. A method for enriching a sample for a plurality ofstructurally different target polynucleotides comprising an immune genesequence the method comprising: a) contacting a sample with a pluralityof immune cell receptor V gene specific primers, each primer includingfrom 5′ to 3′: [5′-Phos], [SPLINT1], [BARCODE], and [V], wherein:[5′-Phos] is a 5′ phosphate; [SPLINT] is a first adaptor sequence;[BARCODE] is a unique molecular identifier barcode; and [V] is asequence capable of hybridizing to an immune cell receptor V gene; b)hybridizing and extending the V gene specific primers to form aplurality of first double-stranded primer extension products; c)contacting the sample with an exonuclease to remove unhybridized V genespecific primers from the first double stranded primer extensionproducts; d) contacting the sample with a plurality of immune cellreceptor J gene specific primers, each primer including from 5′ to 3′:[5′-Phos], [SPLINT2], and [J], wherein: [5′-Phos] is a 5′ phosphate;[SPLINT2] is a second adaptor sequence; and [J] is a sequence capable ofhybridizing to an immune cell receptor J gene; and further contactingthe sample with a first universal primer capable of hybridizing to thefirst adaptor sequence; e) hybridizing and extending the J gene specificprimers and the first universal primer to form a plurality of seconddouble-stranded primer extension products; f) contacting the sample withan exonuclease to remove unhybridized J gene specific primers and firstuniversal primer from the second double-stranded primer extensionproducts; g) contacting the sample with first and second universalprimers capable of hybridizing to the first and second adaptorsequences; h) amplifying the plurality of second double-stranded primerextension products thereby enriching the plurality of structurallydifferent target polynucleotides comprising an immune gene sequence. 2.The method of claim 1, wherein the immune genes comprise one or more ofT-cell receptor alpha (TCRA), T-cell receptor beta (TCRB), T-cellreceptor gamma (TCRG), T-cell receptor delta (TCRD), Immunoglobulinheavy chain (IGH), Immunoglobulin light chain-kappa (IGK) andImmunoglobulin light lambda (IGL).
 3. The method of claim 1, wherein theplurality of V gene specific primers and the plurality of J genespecific primers include primers from Table 2a.
 4. The method of claim1, wherein hybridizing in steps b) and/or e) comprises one or morecycles of a step-wise temperature drop of two or more steps.
 5. Themethod of claim 4, wherein hybridizing in steps b) and/or e) comprises20 cycles of temperature change from 60° C. to 57.5° C. and to 55° C. 6.The method of claim 1, wherein hybridizing and extending in step e)comprises two or more cycles of duplex denaturation, primer annealingand primer extension.
 7. The method of claim 6, wherein primer annealingcomprises one or more cycles of a step-wise temperature drop of two ormore steps.
 8. The method of claim 7, wherein hybridizing and extendingin step e) comprises 10 cycles temperature change from >90° C., to 60°C. to 57.5° C., to 55° C. and to 72° C.
 9. The method of claim 1,wherein the first and second universal primers in step g) compriseadditional 5′sequences not present in the first and second adaptors. 10.The method of claim 9, wherein the first universal primer in step d)does not comprise additional 5′sequences not present in the firstadaptor.
 11. The method of claim 1, wherein the exonuclease in steps c)and/or f) is thermolabile.
 12. The method of claim 11, wherein theexonuclease is Exonuclease I.
 13. The method of claim 1, whereinextending in steps b) and/or e) is with a high-fidelity DNA polymerase.14. The method of claim 1, further comprising a purification step aftersteps c) and/or h).
 15. The method of claim 1, not having a purificationstep between steps f) and g).
 16. The method of claim 15, wherein thefirst and second universal primers comprise a modification preventingdigestion of the primers with the exonuclease.
 17. The method of claim16, wherein the primers comprise one or more nucleotides having amodification selected from: phosphorothioate (PS) bond, a 2′-O-methyl(2′OMe), a 2′-fluoride and Inverted ddT.
 18. The method of claim 1,wherein the V-gene specific primers, the J-gene specific primers and thefirst universal primer in step d) do not comprise a modificationpreventing digestion of the primers with the exonuclease.
 19. The methodof claim 1, further comprising sequencing the plurality of structurallydifferent target polynucleotides comprising an immune gene sequence. 20.A method for contamination-reduced sequencing a plurality ofstructurally different target polynucleotides comprising an immune genesequence the method comprising: a) contacting a sample with a pluralityof immune cell receptor V gene specific primers, each primer includingfrom 5′ to 3′: [5′-Phos], [SPLINT1], [BARCODE], and [V], wherein:[5′-Phos] is a 5′ phosphate; [SPLINT] is a first adaptor sequence;[BARCODE] is a unique molecular identifier barcode (UMI); and [V] is asequence capable of hybridizing to an immune cell receptor V gene; b)hybridizing and extending the V gene specific primers to form aplurality of first double-stranded primer extension products; c)contacting the sample with an exonuclease to remove unhybridized V genespecific primers from the first double stranded primer extensionproducts; d) contacting the sample with a plurality of immune cellreceptor J gene specific primers, each primer including from 5′ to 3′:[5′-Phos], [SPLINT2], and [J], wherein: [5′-Phos] is a 5′ phosphate;[SPLINT2] is a second adaptor sequence; and [J] is a sequence capable ofhybridizing to an immune cell receptor J gene; and further contactingthe sample with a first universal primer capable of hybridizing to thefirst adaptor sequence; e) hybridizing and extending the J gene specificprimers and the first universal primer to form a plurality of seconddouble-stranded primer extension products; f) contacting the sample withan exonuclease to remove unhybridized J gene specific primers and firstuniversal primer from the second double-stranded primer extensionproducts; g) contacting the sample with first and second universalprimers capable of hybridizing to the first and second adaptorsequences; h) amplifying the plurality of second double-stranded primerextension products thereby enriching the plurality of structurallydifferent target polynucleotides comprising an immune gene sequence; i)sequencing the plurality of structurally different targetpolynucleotides comprising an immune gene sequence to obtain a datasetof sequence reads; j) grouping the sequence reads having an identicalUMI into UMI families; k) removing from the dataset UMI families withrelative representation of less than 10%.
 21. The method of claim 20,wherein representation in step k) is less than 9%, 8%, 7%, 6%, 5%, 4%,3%, 2% or 1%.
 22. The method of claim 20, wherein the immune genescomprise one or more of T-cell receptor alpha (TCRA), T-cell receptorbeta (TCRB), T-cell receptor gamma (TCRG), T-cell receptor delta (TCRD),Immunoglobulin heavy chain (IGH), Immunoglobulin light chain-kappa (IGK)and Immunoglobulin light chain-lambda (IGL).
 23. The method of claim 20,wherein the plurality of V gene specific primers and the plurality of Jgene specific primers include primers from Table 2a.
 24. The method ofclaim 20, wherein hybridizing in steps b) and/or e) comprises one ormore cycles of a step-wise temperature drop of two or more steps. 25.The method of claim 24, wherein hybridizing in steps b) and/or e)comprises 20 cycles of temperature change from 60° C. to 57.5° C. and to55° C.
 26. The method of claim 20, wherein hybridizing and extending instep e) comprises two or more cycles of duplex denaturation, primerannealing and primer extension.
 27. The method of claim 26, whereinprimer annealing comprises one or more cycles of a step-wise temperaturedrop of two or more steps.
 28. The method of claim 27, whereinhybridizing and extending in step e) comprises 10 cycles temperaturechange from >90° C., to 60° C. to 57.5° C., to 55° C. and to 72° C. 29.The method of claim 20, wherein the first and second universal primersin step g) comprise additional 5′sequences not present in the first andsecond adaptors.
 30. The method of claim 29, wherein the first universalprimer in step d) does not comprise additional 5′sequences not presentin the first adaptor.
 31. The method of claim 20, wherein theexonuclease in steps c) and/or f) is thermolabile.
 32. The method ofclaim 31, wherein the exonuclease is Exonuclease I.
 33. The method ofclaim 20, wherein extending in steps b) and/or e) is with ahigh-fidelity DNA polymerase.
 34. The method of claim 20, furthercomprising a purification step after steps c) and/or h).
 35. The methodof claim 20, not having a purification step between steps f) and g). 36.The method of claim 35, wherein the first and second universal primerscomprise a modification preventing digestion of the primers with theexonuclease.
 37. The method of claim 36, wherein the primers compriseone or more nucleotides having a modification selected from:phosphorothioate (PS) bond, a 2′-O-methyl (2′OMe), a 2′-fluoride andInverted ddT.
 38. The method of claim 20, wherein the V-gene specificprimers, the J-gene specific primers and the first universal primer instep d) do not comprise a modification preventing digestion of theprimers with the exonuclease.